Bacteria-based protein delivery

ABSTRACT

The present invention relates to recombinant Gram-negative bacterial strains and the use thereof for delivery of repeated domains of a heterologous protein or two or more domains of different heterologous proteins into eukaryotic cells.

INCORPORATION OF SEQUENCE LISTING

The sequence listing named “LATS-006 Seq List_July 23 2020_ST25” whichwas created on Jul. 23, 2020 and is 251 KB in size, is herebyincorporated by reference in its entirety.

THE FIELD OF THE INVENTION

The present invention relates to recombinant Gram-negative bacterialstrains and the use thereof for delivery of repeated domains of aheterologous protein or two or more domains of different heterologousproteins into eukaryotic cells.

BACKGROUND OF THE INVENTION

Transient transfection techniques have been applied in cell biologicalresearch over many years to address protein functions. These methodsgenerally result in a massive overrepresentation of the protein understudy, which might lead to oversimplified models of signalling. Forproteins controlling short-lived signalling processes, the protein ofinterest is present for far longer as the signalling event it controls.Even more, DNA transfection based transient over-expression leads to aheterogenous and unsynchronized cell population, which complicatesfunctional studies and hampers—omics approaches. Besides this, theupscaling of such assays to a larger scale is very expensive. Some ofthe above mentioned points are covered by existing techniques asmicroinjection or proteo-fection of purified proteins, the inducibletranslocation strategy to rapidly target plasmid born small GTPases tothe cell membrane or the addition of purified proteins fused tocell-permeable bacterial toxins. But these techniques are alltime-consuming and cumbersome and to our knowledge none fulfils allmentioned criteria.

Bacteria have evolved different mechanisms to directly inject proteinsinto target cells [1]. The type III secretion system (T3SS) used bybacteria like Yersinia, Shigella and Salmonella [2] functions like anano-syringe that injects so-called bacterial effector proteins intohost cells. Bacterial proteins to be secreted via the T3SS, calledeffectors, harbour a short N-terminal secretion signal [3]. Insidebacteria, some effectors are bound by chaperones. Chaperones might masktoxic domains, they contribute to exposition of the secretion signal andkeep the substrates in a secretion-competent conformation, thereforefacilitating secretion. Upon induction of secretion, an ATPase adjacentto the T3SS removes the chaperones and the effectors travel unfolded oronly partially folded through the needle, and refold once in the hostcytoplasm.

T3S has been exploited to deliver hybrid peptides and proteins intotarget cells. Heterologous bacterial T3SS effectors have been deliveredin case the bacterium under study is hardly accessible by genetics (likeChlamydia trachomatis). Often reporter proteins were fused to possibleT3SS secretion signals as to study requirements for T3SS dependentprotein delivery, such as the Bordetella pertussis adenylate cyclase,murine DHFR or a phosphorylatable tag. Peptide delivery was mainlyconducted with the aim of vaccination. This includes viral epitopes,bacterial epitopes (listeriolysin O) as well as peptides representingepitopes of human cancer cells. In few cases functional eukaryoticproteins have been delivered to modulate the host cell, as done withnanobodies [4], nuclear proteins (Cre-recombinase, MyoD) [5,6] or Il10and IL1ra [7]. None of the above-mentioned systems allows single-proteindelivery as in each case one or multiple endogenous effector proteinsare still encoded. Furthermore, the vectors used have not been designedin a way allowing simple cloning of other DNA fragments encodingproteins of choice, hindering broad application of the system.Surprisingly it has been found that delivery of repeated domains ofheterologous proteins or combinations of domains of differentheterologous proteins to eukaryotic cells enlarges the impact on adesired cellular pathway.

SUMMARY OF THE INVENTION

The present invention relates generally to recombinant Gram-negativebacterial strains and the use thereof for delivery of repeated domainsof a heterologous protein or two or more domains of differentheterologous proteins into eukaryotic cells. The present inventionprovides Gram-negative bacterial strains and the use thereof, whichallows the translocation of repeated domains of a heterologous proteinor two or more domains of different heterologous proteins such asvarious type III effectors, but also of type IV effectors, viralproteins and most importantly functional eukaryotic proteins. Means forfluorescent tracking of delivery, for relocalization to the nucleus andnotably for removal of the bacterial appendage after delivery to thehost cell are provided. The presented T3SS based system results inscalable, rapid, synchronized, homogenous and tunable delivery ofrepeated domains of a heterologous protein or two or more domains ofdifferent heterologous proteins of interest. The delivery system of thepresent invention is suitable to inject repeated domains of a eukaryoticprotein or two or more domains of different eukaryotic proteins inliving animals and can be used for therapeutic purposes.

In a first aspect the present invention relates to a recombinantGram-negative bacterial strain transformed with a vector which comprisesin the 5′ to 3′ direction:

a promoter;

a first DNA sequence encoding a delivery signal from a bacterial T3SSeffector protein, operably linked to said promoter; and

a second DNA sequence encoding repeated domains of a heterologousprotein or two or more domains of different heterologous proteins fusedin frame to the 3′ end of said first DNA sequence, wherein theheterologous proteins are selected from the group consisting of proteinsinvolved in apoptosis or apoptosis regulation, cell cycle regulators,ankyrin repeat proteins, cell signaling proteins, reporter proteins,transcription factors, proteases, small GTPases, GPCR related proteins,nanobody fusion constructs and nanobodies, bacterial T3SS effectors,bacterial T4SS effectors and viral proteins.

In a further aspect the present invention relates to a recombinantGram-negative bacterial strain transformed with a vector which comprisesin the 5′ to 3′ direction:

a first DNA sequence encoding a delivery signal or a fragment thereoffrom a bacterial effector protein; and

a second DNA sequence encoding repeated domains of a heterologousprotein or two or more domains of different heterologous proteins fusedin frame to the 3′ end of said first DNA sequence, wherein theheterologous proteins are selected from the group consisting of proteinsinvolved in apoptosis or apoptosis regulation, cell cycle regulators,ankyrin repeat proteins, cell signaling proteins, reporter proteins,transcription factors, proteases, small GTPases, GPCR related proteins,nanobody fusion constructs and nanobodies, bacterial T3SS effectors,bacterial T4SS effectors and viral proteins.

In a further aspect the present invention relates to a vector whichcomprises in the 5′ to 3′ direction:

a first DNA sequence encoding a delivery signal or a fragment thereoffrom a bacterial effector protein; and

a second DNA sequence encoding repeated domains of a heterologousprotein or two or more domains of different heterologous proteins fusedin frame to the 3′ end of said first DNA sequence, wherein theheterologous proteins are selected from the group consisting of proteinsinvolved in apoptosis or apoptosis regulation, cell cycle regulators,ankyrin repeat proteins, cell signaling proteins, reporter proteins,transcription factors, proteases, small GTPases, GPCR related proteins,nanobody fusion constructs and nanobodies, bacterial T3SS effectors,bacterial T4SS effectors and viral proteins.

In a further aspect the present invention relates to a vector whichcomprises in the 5′ to 3′ direction:

a promoter;

a first DNA sequence encoding a delivery signal from a bacterial T3SSeffector protein, operably linked to said promoter;

a second DNA sequence encoding repeated domains of a heterologousprotein or two or more domains of different heterologous proteins fusedin frame to the 3′ end of said first DNA sequence,

wherein the heterologous proteins are selected from the group consistingof proteins involved in apoptosis or apoptosis regulation, cell cycleregulators, ankyrin repeat proteins, cell signaling proteins, reporterproteins, transcription factors, proteases, small GTPases, GPCR relatedproteins, nanobody fusion constructs and nanobodies, bacterial T3SSeffectors, bacterial T4SS effectors and viral proteins.

The present invention further relates to a method for deliveringrepeated domains of a heterologous protein or two or more domains ofdifferent heterologous proteins into a eukaryotic cell comprising thefollowing steps:

i) culturing a Gram-negative bacterial strain; and

ii) contacting a eukaryotic cell with the Gram-negative bacterial strainof i) wherein a fusion protein which comprises a delivery signal from abacterial T3SS effector protein and the repeated domains of aheterologous protein or two or more domains of different heterologousproteins is expressed by the Gram-negative bacterial strain and istranslocated into the eukaryotic cell.

The present invention further relates to a method for deliveringrepeated domains of a heterologous protein or two or more domains ofdifferent heterologous proteins into a eukaryotic cell comprising thefollowing steps:

i) culturing a Gram-negative bacterial strain;

ii) contacting a eukaryotic cell with the Gram-negative bacterial strainof i) wherein a fusion protein which comprises a delivery signal from abacterial T3SS effector protein and the repeated domains of aheterologous protein or two or more domains of different heterologousproteins is expressed by the Gram-negative bacterial strain and istranslocated into the eukaryotic cell; andiii) cleaving the fusion protein so that the repeated domains of aheterologous protein or the two or more domains of differentheterologous proteins are cleaved from the delivery signal from thebacterial T3SS effector protein.

In a further aspect the present invention relates to a library ofGram-negative bacterial strains, wherein the repeated domains of aheterologous protein or the two or more domains of differentheterologous proteins encoded by the second DNA sequence of theexpression vector of the Gram-negative bacterial strains are domains ofa human or murine protein and, wherein each domain of a human or murineprotein expressed by a Gram-negative bacterial strain is different inamino acid sequence.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 : Characterization of T3SS protein delivery. (A) Schematicrepresentation of T3SS dependent protein secretion into the surroundingmedium (in-vitro secretion)(left side) or into eukaryotic cells (rightside). I: shows the type 3 secretion system. II indicates proteinssecreted into the surrounding medium, III proteins translocated throughthe membrane into the cytosol of eukaryotic cells (VII). VI shows astretch of the two bacterial membranes in which the T3SS is inserted andthe bacterial cytosol underneath. IV is a fusion protein attached to theYopE₁₋₁₃₈ N-terminal fragment (V) (B) In-vitro secretion of I: Y.enterocolitica E40 wild type, II: Y. enterocolitica ΔHOPEMT asd or III:Y. enterocolitica ΔHOPEMT asd+pBadSi_2 as revealed by Western blottingon total bacterial lysates (IV) and precipitated culture supernatants(V) using an anti-YopE antibody.

FIG. 2 : Characterization of T3SS protein delivery into epithelialcells. (A) Anti-Myc immunofluorescence staining on HeLa cells infectedat an MOI of 100 for 1 h with I: Y. enterocolitica ΔHOPEMT asd or II: Y.enterocolitica ΔHOPEMT asd+pBad_Si2. (B) Quantification of anti-Mycimmunofluorescence staining intensity from (A) within HeLa cells. Datawere combined from n=20 sites, error bars indicated are standard errorof the mean. I: uninfected, II: Y. enterocolitica ΔHOPEMT asd or III: Y.enterocolitica ΔHOPEMT asd+pBad_Si2. Y-axis indicates anti-Myc stainingintensity [arbitrary unit], x-axis indicates time of infection inminutes (C) Quantification of Anti-Myc immunofluorescence stainingintensity within cells. HeLa cells were infected for 1 h with Y.enterocolitica ΔHOPEMT asd+pBad_Si2 at an MOI indicated on the x-axis.Data were combined from n=20 sites, error bars indicated are standarderror of the mean. Y-axis indicates anti-Myc staining intensity [a.u.].

FIG. 3 : Modifications of the T3SS based protein delivery allow nuclearlocalization of a YopE₁₋₁₃₈ fusion protein (EGFP). EGFP signal in HeLacells infected with I: Y. enterocolitica ΔHOPEMT asd or II: Y.enterocolitica ΔHOPEMT asd ΔyopB carrying the plasmids III:+YopE₁₋₁₃₈-EGFP or IV: +YopE₁₋₁₃₈-EGFP-NLS at an MOI of 100. EGFP signalis shown in “a”, for localization comparison nuclei were stained in “b”.

FIG. 4 : Modifications of the T3SS based protein delivery allow removalof the YopE₁₋₁₃₈ appendage. HeLa cells are infected with two differentY. enterocolitica strains at the same time, which is reached by simplemixing of the two bacterial suspensions. One strain is delivering theTEV protease fused to YopE₁₋₁₃₈, while the other strain delivers aprotein of interest fused to YopE₁₋₁₃₈ with a linker containing a doubleTEV protease cleavage site. After protein delivery into the eukaryoticcell, the TEV protease will cleave the YopE₁₋₁₃₈ appendage from theprotein of interest (A) Digitonin lysed HeLa cells uninfected (II) orafter infection (MOI of 100) for 2 h with I: Y. enterocolitica ΔHOPEMTasd and III: +pBadSi_2, IV: +YopE₁₋₁₃₈-2×TEV cleavage site-Flag-INK4C,V: +YopE₁₋₁₃₈-2×TEV cleavage site-Flag-INK4C and further overnighttreatment with purified TEV protease and VI: +YopE₁₋₁₃₈-2×TEV cleavagesite-Flag-INK4C and a second strain+YopE₁₋₁₃₈-TEV were analyzed byWestern blotting anti-INK4C (shown in “a”) for the presence ofYopE₁₋₁₃₈-2×TEV cleavage site-Flag-INK4C or its cleaved form Flag-INK4C.As a loading control western blotting anti-Actin was performed (shown in“b”). In one case (V) the lysed cells were incubated overnight withpurified TEV protease. (B) Actin normalized quantification of anti-INK4Cstaining intensity (shown as [a.u.] on the y-axis) from (A) at the sizeof full length YopE₁₋₁₃₈-2×TEV cleavage site-Flag-INK4C, where sample IVis set to 100%. I: Y. enterocolitica ΔHOPEMT asd and IV: +YopE₁₋₁₃₈-2×TEV cleavage site-Flag-INK4C, V: +YopE₁₋₁₃₈-2×TEV cleavagesite-Flag-INK4C and further overnight treatment with purified TEVprotease and VI: +YopE₁₋₁₃₈-2×TEV cleavage site-Flag-INK4C and a secondstrain+YopE₁₋₁₃₈-TEV. Data were combined from n=2 independentexperiments, error bars indicated are standard error of the mean (C)Digitonin lysed HeLa cells uninfected (II) or after infection (MOI of100) for 2 h with I: Y. enterocolitica ΔHOPEMT asd and III: +pBadSi_2,IV: +YopE₁₋₁₃₈-2×TEV cleavage site-ET1-Myc, V: +YopE₁₋₁₃₈-2×TEV cleavagesite-ET1-Myc and further overnight treatment with purified TEV proteaseand VI: +YopE₁₋₁₃₈-2×TEV cleavage site-ET1-Myc and a secondstrain+YopE₁₋₁₃₈-TEV were analyzed by Western blotting anti-Myc (shownin “a”) for the presence of YopE₁₋₁₃₈-2×TEV cleavage site-ET1-Myc or itscleaved form ET1-Myc. As a loading control western blotting anti-Actinwas performed (shown in “b”) In one case (V) the lysed cells wereincubated overnight with purified TEV protease.

FIG. 5 : Delivery of bacterial effector proteins into eukaryotic cells(A) HeLa cells were infected with I: Y. enterocolitica ΔHOPEMT asdcarrying II: pBad_Si2 or III: YopE₁₋₁₃₈-SopE at an MOI of 100 for thetime indicated above the images (2, 10 or 60 minutes). After fixationcells were stained for the actin cytoskeleton (B) HeLa cells were leftuninfected (II) or infected with I: Y. enterocolitica ΔHOPEMT asdcarrying III: YopE₁₋₁₃₈-SopE-Myc and in some cases coinfected with IV:YopE₁₋₁₃₈-SptP at the MOI indicated below the strain (MOI 50;MOI50:MOI50 or MOI50:MOI100) for 1 h. After fixation cells were stainedfor the actin cytoskeleton (shown in “a”) and the presence of theYopE₁₋₁₃₈-SopE-Myc fusion protein was followed via staining anti-Myc(shown in “b”).

FIG. 6 : Delivery of bacterial effector proteins into eukaryotic cells(A) Phospho-p38 (“a”), total p38 (“b”) and actin (“c”) western blotanalysis on HeLa cells left untreated (II) or infected for 75 min withI: Y. enterocolitica ΔHOPEMT asd carrying III: pBad_Si2 or IV:YopE₁₋₁₃₈-OspF at an MOI of 100. Cells were stimulated with TNFα for thelast 30 min of the infection as indicated (+ stands for addition ofTNFα, − represent no treatment with TNFα) (B) Phospho-Akt T308 (“a”) andS473 (“b”) and actin (“c”) western blot analysis on HeLa cells leftuntreated (II) or infected for 22.5 or 45 min (indicated below theblots) with I: Y. enterocolitica ΔHOPEMT asd carrying III: pBad_Si2, IV:YopE₁₋₁₃₈-SopE or V: YopE₁₋₁₃₈-SopB at an MOI of 100 (C) cAMP levels (infmol/well shown on y-axis) in HeLa cells left untreated (I) or infectedfor 2.5 h with V: Y. enterocolitica ΔHOPEMT asd+YopE ₁₋₁₃₈-BepA, VI: Y.enterocolitica ΔHOPEMT asd+YopE ₁₋₁₃₈-BepA_(E305-end), VII: Y.enterocolitica ΔHOPEMT asd+YopE ₁₋₁₃₈-BepG_(Bid) or VIII: Y.enterocolitica ΔHOPEMT asd+pBad_Si2 at an MOI of 100. Cholera toxin (CT)was added for 1 h as positive control to samples II (1 μg/ml), III (25μg/ml) or IV (50 μg/ml). Data were combined from n=3 independentexperiments, error bars indicated are standard error of the mean.Statistical analysis was performed using an unpaired two-tailed t-test(ns indicates a non significant change, ** indicates a p value <0.01,*** indicates a p value <0.001).

FIG. 7 : Delivery of human tBid into eukaryotic cells induces massiveapoptosis. (A) Cleaved Caspase 3 p17 (“a”) and actin (“b”) western blotanalysis on HeLa cells left untreated (II) or infected for 60 min withI: Y. enterocolitica ΔHOPEMT asd carrying III: pBad_Si2, IV:YopE₁₋₁₃₈-Bid or V: YopE₁₋₁₃₈-t-Bid at an MOI of 100. In some cases,cells were treated with VI: 0.5 μM Staurosporine or VII: 1 μMStaurosporine (B) Digitonin lysed HeLa cells left untreated (II) orafter infection for 1 h with I: Y. enterocolitica ΔHOPEMT asd carryingIII: pBad_Si2, IV: YopE₁₋₁₃₈-Bid or V: YopE₁₋₁₃₈-t-Bid at an MOI of 100were analyzed by Western blotting anti-Bid (“a”) allowing comparison ofendogenous Bid levels (marked Z) to translocated YopE₁₋₁₃₈-Bid (markedX) or YopE₁₋₁₃₈-tBid (marked Y) levels. As a loading control westernblotting anti-Actin was performed (shown in “b”). In some cases, cellswere treated with VI: 0.5 μM Staurosporine or VII: 1 μM Staurosporine(C) HeLa cells were left untreated (I) or infected at an MOI of 100 for1 h with II: Y. enterocolitica ΔHOPEMT asd+pBad_Si2, III: Y.enterocolitica ΔHOPEMT asd+YopE₁₋₁₃₈-Bid, IV: Y. enterocolitica ΔHOPEMTasd+YopE ₁₋₁₃₈-tBid. In some cases, cells were treated with V: 0.5 μMStaurosporine or VI: 1 μM Staurosporine. After fixation cells werestained for the actin cytoskeleton (gray).

FIG. 8 : T3SS dependent delivery of zebrafish BIM induces apoptosis inzebrafish embryos. (A) 2 dpf zebrafish embryos were infected with theEGFP expressing Y. enterocolitica ΔHOPEMT asd+pBad_Si1 control strain(I) or zBIM translocating strain (II: Y. enterocolitica ΔHOPEMTasd+YopE₁₋₁₃₈-zBIM) by injection of about 400 bacteria into thehindbrain region. After 5.5 h the embryos were fixed, stained foractivated Caspase 3 (cleaved Caspase 3, p17; shown in “c”) and analyzedfor presence of bacteria (EGFP signal, shown in “b”). Maximum intensityz projections are shown for fluorescent images. Bright-field zprojection are shown in “a” (B) Automated image analysis on maximumintensity z projections of recorded z-stack images of (A). Briefly,bacteria were detected via the GFP channel. Around each area of abacterial spot a circle with a radius of 10 pixels was created.Overlapping regions were separated equally among the connecting members.In those areas closely surrounding bacteria, the Caspase 3 p17 stainingintensity was measured and is plotted on the y-axis (as [a.u.]).Statistical analysis was performed using a Mann-Whitney test (***indicates a p value <0.001). Data were combined from n=14 for Y.enterocolitica ΔHOPEMT asd+pBad_Si1 control strain (I) or n=19 for II:Y. enterocolitica ΔHOPEMT asd+YopE₁₋₁₃₈-zBIM infected animals, errorbars indicated are standard error of the mean.

FIG. 9 : tBiD dependent phosphoproteome: HeLa cells were infected for 30min with Y. enterocolitica ΔHOPEMT asd+YopE₁₋₁₃₈-t-Bid at an MOI of 100and as a control with Y. enterocolitica ΔHOPEMT asd+pBad_Si2. (A)Graphical representation of the tBID phosphoproteome. Proteinscontaining phosphopeptides that were significantly regulated in a tBiddependent manner (gray) (q-value <0.01) as well as known apopotosisrelated proteins (dark gray) are represented in a STRING network ofknown and predicted protein-protein interactions (high-confidence, score0.7). Only proteins with at least one connection in STRING arerepresented. (B) Confocal images of HeLa cells infected with either Y.enterocolitica ΔHOPEMT asd+pBad_Si2 (I) or Y. enterocolitica ΔHOPEMTasd+YopE₁₋₁₃₈-t-Bid (II) reveal the induction of an apoptotic phenotypeupon tBid delivery. Cells were stained for the nuclei with Hoechst(“a”), for F-actin with phalloidin (“b”), for tubulin with ananti-tubulin antibody (“c”) and for mitochondria with mitotracker (“d)”.Scale bar represents 40 μm.

FIG. 10 : Description of the type III secretion-based delivery toolbox.(A) Vector maps of the cloning plasmids pBad_Si1 and pBad_Si2 used togenerate fusion constructs with YopE₁₋₁₃₈. The chaperone SycE and theYopE₁₋₁₃₈-fusion are under the native Y. enterocolitica promoter. Thetwo plasmids only differ in presence of an arabinose inducible EGFPpresent on pBad_Si1 (B) Multiple cloning site directly following theyopE₁₋₁₃₈ fragment on pBad_Si1 and pBad_Si2 plasmids. The sequence isset forth in SEQ ID NO: 225.

FIG. 11 : Characterization of T3SS protein delivery into various celllines. Anti-Myc immunofluorescence staining on Swiss 3T3 fibroblasts(“a”), Jurkat cells (“b”) and HUVEC cells (“c”) left untreated (II) orinfected with Y. enterocolitica ΔHOPEMT asd+pBad_Si2 (I) at the MOIindicated above the images (MOI 25, 50, 100, 200 and 400 for HUVECs) for1 h.

FIG. 12 : T3SS dependency of delivery of bacterial effector proteinsinto eukaryotic cell. Digitonin lysed HeLa cells after infection at anMOI of 100 for time indicated above the blots (0, 5, 15, 10, 60 and 120minutes) with Y. enterocolitica ΔHOPEMT asd ΔyopB+YopE ₁₋₁₃₈-SopE-Myc(I) or Y. enterocolitica ΔHOPEMT asd+YopE₁₋₁₃₈-SopE-Myc (II) wereanalyzed by Western blotting anti-Myc. The size corresponding toYopE₁₋₁₃₈-SopE-Myc is marked with “a”, while the size of the endogenousc-Myc protein is marked with “b”.

FIGS. 13 and 14 : T3SS dependent secretion of various other proteinsinto the culture supernatant. In-vitro secretion experiment of I: Y.enterocolitica ΔHOPEMT asd+YopE ₁₋₁₃₈ fused to the protein as indicated.Protein content of total bacterial lysates (“A”) and precipitatedculture supernatants (“B”) was analyzed by Western blotting using ananti-YopE antibody. Numbers written indicate molecular weight in kDa atthe corresponding height.

FIGS. 15A to N: Y. enterocolitica and S. enterica strains used in thisstudy.

List of Y. enterocolitica and S. enterica strains used in this studyproviding information on background strains, plasmids and proteins forT3SS dependent delivery encoded on corresponding plasmids. Further,information on oligonucleotides used for construction of thecorresponding plasmid, the backbone plasmid and antibiotic resistancesis provided.

FIG. 16 : Delivery of murine tBid, murine Bid BH3 and murine Bax BH3into B16F10 cells induces massive apoptosis. B16F10 cells uninfected (I)or after infection (MOI of 50) for 2.5 h with Y. enterocolitica ΔHOPEMTasd and II: +pBadSi_2, III: +YopE₁₋₁₃₈-Y. enterocolitica codon optimizedmurine tBid, IV: +YopE₁₋₁₃₈-Y. enterocolitica codon optimized murine BidBH3 or V: +YopE₁₋₁₃₈-Y. enterocolitica codon optimized murine Bax BH3.After fixation cells were stained for the actin cytoskeleton and nuclei(both in gray).

FIG. 17 : Delivery of murine tBid, murine Bid BH3 and murine Bax BH3into D2A1 cells induces massive apoptosis. D2A1 cells uninfected (I) orafter infection (MOI of 50) for 2.5 h with Y. enterocolitica ΔHOPEMT asdand II: +pBadSi 2, III: +YopE₁₋₁₃₈-Y. enterocolitica codon optimizedmurine tBid, IV: +YopE₁₋₁₃₈-Y. enterocolitica codon optimized murine BidBH3 or V: +YopE₁₋₁₃₈-Y. enterocolitica codon optimized murine Bax BH3.After fixation cells were stained for the actin cytoskeleton and nuclei(both in gray).

FIG. 18 : Delivery of murine tBid, murine Bid BH3 and murine Bax BH3into HeLa cells induces massive apoptosis. HeLa cells uninfected (I) orafter infection (MOI of 50) for 2.5 h with Y. enterocolitica ΔHOPEMT asdand II: +pBadSi 2, III: +YopE₁₋₁₃₈-Y. enterocolitica codon optimizedmurine tBid, IV: +YopE₁₋₁₃₈-Y. enterocolitica codon optimized murine BidBH3 or V: +YopE₁₋₁₃₈-Y. enterocolitica codon optimized murine Bax BH3.After fixation cells were stained for the actin cytoskeleton and nuclei(both in gray).

FIG. 19 : Delivery of murine tBid, murine Bid BH3 and murine Bax BH3into 4T1 cells induces massive apoptosis. 4T1 cells uninfected (I) orafter infection (MOI of 50) for 2.5 h with Y. enterocolitica ΔHOPEMT asdand II: +pBadSi_2, III: +YopE₁₋₁₃₈-Y. enterocolitica codon optimizedmurine tBid, IV: +YopE₁₋₁₃₈-Y. enterocolitica codon optimized murine BidBH3 or V: +YopE₁₋₁₃₈-Y. enterocolitica codon optimized murine Bax BH3.After fixation cells were stained for the actin cytoskeleton and nuclei(both in gray).

FIG. 20 : Delivery of murine tBid by S. enterica grown under SPI-1 T3SSinducing conditions into eukaryotic cells induces apoptosis. CleavedCaspase 3 p 17 western blot analysis on HeLa cells left untreated (I) orinfected for 4 h with III: S. enterica aroA carrying IV: SteA₁₋₂₀-t-Bid,V: SteA_(FL)-Bid, VI: SopE₁₋₈₁-t-Bid or VII: SopE₁₋₁₀₅-t-Bid at an MOIof 100. For this experiment, all S. enterica aroA strains were grownunder SPI-1 T3SS inducing conditions. In some cases, cells were treatedwith II: 1 μM Staurosporine. Numbers written indicate molecular weightin kDa at the corresponding height.

FIG. 21 : Delivery of murine tBid by S. enterica grown under SPI-2 T3SSinducing conditions into eukaryotic cells induces apoptosis. CleavedCaspase 3 p17 western blot analysis on HeLa cells left untreated (I) orinfected for 4 h with III: S. enterica aroA carrying IV: SteA₁₋₂₀-t-Bid,V: SteA_(FL)-Bid, VI: SopE₁₋₈₁-t-Bid or VII: SopE₁₋₁₀₅-t-Bid at an MOIof 100. For this experiment, all S. enterica aroA strains were grownunder SPI-2 T3SS inducing conditions. In some cases, cells were treatedwith II: 1 μM Staurosporine. Numbers written indicate molecular weightin kDa at the corresponding height.

FIG. 22 : S. enterica T3SS dependent secretion of various cell cycleproteins into the culture supernatant. In-vitro secretion experiment ofS. enterica aroA+either SteA_(FL) (I, III, V, VII) or SopE₁₋₁₀₅ (II, IV,VI, VIII) fused to proteins as listed following. I and II: Ink4a-MycHis;III and IV: Ink4c-MycHis; V and VI: Mad2-MycHis; VII and VIII:Cdk1-MycHis. Protein content of precipitated culture supernatants (“A”)and total bacterial lysates (“B”) was analyzed by Western blotting usingan anti-myc antibody. Numbers written indicate molecular weight in kDaat the corresponding height.

FIG. 23 : T3SS dependent secretion of various known cell cycleinterfering peptides into the culture supernatant. In-vitro secretionexperiment of I: Y. enterocolitica ΔHOPEMT asd+pBad_Si2. II-VII: Y.enterocolitica ΔHOPEMT asd+YopE ₁₋₁₃₈ fused to peptides as listedfollowing: II: Ink4A₈₄₋₁₀₃; III: p107/RBL1₆₅₇₋₆₆₂; IV:p21_(141-160D149A); V: p21_(145-160D149A); p21₁₇₋₃₃; VII: cyclinD2₁₃₉₋₁₄₇. Protein content of precipitated culture supernatants (“A”)and total bacterial lysates (“B”) was analyzed by Western blotting usingan anti-YopE antibody. Numbers written indicate molecular weight in kDaat the corresponding height.

FIG. 24 : Fusion of the T3SS delivered protein to Ubiquitin allowsremoval of the YopE₁₋₁₃₈ appendage. HeLa cells are infected with astrain delivering a protein of interest fused to YopE₁₋₁₃₈ with adirectly fused Ubiquitin (YopE₁₋₁₃₈-Ubi). After protein delivery intothe eukaryotic cell, endogenous Ubiquitin specific proteases will cleavethe YopE₁₋₁₃₈-Ubi appendage from the protein of interest. Digitoninlysed HeLa cells uninfected (I) or after infection (MOI of 100) for 1 hwith II: Y. enterocolitica ΔHOPEMT asd+YopE ₁₋₁₃₈-Flag-INK4C-MycHis orIII: +YopE₁₋₁₃₈-Flag-Ubiquitin-INK4C-MycHis were analyzed by Westernblotting anti-INK4C for the presence of IV:YopE₁₋₁₃₈-Flag-Ubiquitin-INK4C-MycHis or V: YopE₁₋₁₃₈-Flag-INK4C-MycHis,the cleaved form VI: INK4C-MycHis and VII: the endogenous INK4C.

FIG. 25 : Schematic representation of heterologous proteins and domainsthereof to be delivered via the bacteria T3SS. I: Human/murinefull-length protein with domains colored in various grayscale, II:Truncated human/murine protein with domains colored in variousgrayscale, III: Motif/domain of human/murine full-length protein only,IV: Motif/domain only repeated (left) or combination of two differentmotifs/domains of human/murine full-length protein (right).

FIG. 26 : Delivery of BH3 domains of murine tBid and murine Bax intoeukaryotic cells and fused repeats thereof induce apoptosis in cancerouscells. B16F10 murine melanoma cells were infected at a MOI of 5, 10, 25and 50 (left to right in each condition) of corresponding bacteria asindicated for 4 h. Effect on cell viability was assessed by countingcell numbers via nuclear counting. I: nuclear count. II: Y.enterocolitica ΔHOPEMT asd+pBad_Si2, III: Y. enterocolitica ΔHOPEMTasd+YopE ₁₋₁₃₈-Bid-BH3, IV: Y. enterocolitica ΔHOPEMT asd+YopE₁₋₁₃₈-(Bid-BH3)₂ and V: Y. enterocolitica ΔHOPEMT asd+YopE₁₋₁₃₈-(Bid-BH3)-(Bax-BH3). Nuclei were stained with Hoechst. Images wereacquired using an automated microscope and cell number was automaticallydetermined using CellProfiler.

FIG. 27 : Delivery of BH3 domains of murine tBid and murine Bax intoeukaryotic cells and fused repeats thereof induce apoptosis in cancerouscells. 4T1 murine breast cancer cells were infected at a MOI of 5, 10,25 and 50 (left to right in each condition) of corresponding bacteria asindicated for 4 h. Effect on cell viability was assessed by countingcell numbers via nuclear counting. I: nuclear count. II: Y.enterocolitica ΔHOPEMT asd+pBad_Si2, III: Y. enterocolitica ΔHOPEMTasd+YopE ₁₋₁₃₈-Bid-BH3, IV: Y. enterocolitica ΔHOPEMT asd+YopE₁₋₁₃₈-(Bid-BH3)₂ and V: Y. enterocolitica ΔHOPEMT asd+YopE₁₋₁₃₈-(Bid-BH3)-(Bax-BH3). Nuclei were stained with Hoechst. Images wereacquired using an automated microscope and cell number was automaticallydetermined using CellProfiler.

FIG. 28 : Delivery of synthetic increased pro-apoptotic proteins.Delivery of single synthetic proteins consisting of single or tandemrepeats of BH3 domains originating from pro-apoptotic proteins t-BID orBAX leads to enhanced apoptosis induction in 4T1 and B16F10 cancerouscells. 4T1 (I) or B16F10 (II) cells were infected with Y. enterocoliticaΔyopHOPEMT encoding on pBad-MycHisA IV: YopE₁₋₁₃₈-tBID BH3 extendeddomain, V: YopE₁₋₁₃₈-linker-tBID BH3, VI: YopE₁₋₁₃₈-tBID BH3, VII:YopE₁₋₁₃₈-(tBID BH3)₂, VIII: YopE₁₋₁₃₈-tBID BH3-BAX BH3 or IX:YopE₁₋₁₃₈-BAX BH3-tBID BH3. A titration of the bacteria added to thecells (MOI) was performed for each strain, cell counts determined andIC50 calculated using non-linear regression. IC50 MOI is indicated(III).

FIG. 29 : Induction of apoptosis by pYV-encoded synthetic pro-apoptoticproteins. Delivery of a single or a tandem repeat of BID BH3 domainencoded on the pYV leads to apoptosis induction in 4T1 and B16F10cancerous cells. 4T1 (I) or B16F10 (II) cells were infected with Y.enterocolitica ΔHOPEMT+IV: pYV-YopE₁₋₁₃₈-BH3-Bid, or V:+pYV-YopE₁₋₁₃₈-(BH3-Bid)₂ or VI: with Y. enterocolitica ΔHOPEMTpBad-MycHisA-YopE₁₋₁₃₈-(BH3-Bid)₂ for 3 hours. A titration of thebacteria added to the cells (MOI) was performed for each strain, cellcounts determined and IC50 (III) calculated using non-linear regression.

FIG. 30 : Tumor colonization of i.v. injected Y. enterocoliticaΔyopH,O,P,E,M,T in the 4T1 breast cancer allograft model. Bacterialcounts in tumors are indicated as colony forming units (CFU) per gram oftissue (III). Counts were assessed in tumors at day 8 (I) and 14 (II)post infection. Each dot represents an individual mouse. The horizontaldashed line indicates the detection limit.

FIG. 31 : Biodistribution of i.v. injected Y. enterocoliticaΔyopH,O,P,E,M,T in the 4T1 breast cancer allograft model. Bacterialcounts in blood (I), spleen (II), liver (III), lung (IV) and tumor (V)are indicated as colony forming units (CFU) per gram of tissue or per mlof blood (VI). Counts were assessed at day 14 post infection. Each dotrepresents an individual mouse. The horizontal dashed line indicates thedetection limit. * indicates a mouse with large metastases found onlung.

FIG. 32 : Delay of tumor progression in wildtype Balb/C mice allografteds.c. with 4T1 breast cancer cells. Wildtype Balb/C mice allografted s.c.with 4T1 breast cancer cells were i.v. injected with I: PBS or II: 1*10⁷Y. enterocolitica dHOPEMT+pYV-YopE₁₋₁₃₈(BH3-Bid)₂, once the tumor hadreached a size of 150-250 mm3. The day of the i.v. injection of bacteriawas defined as day 0. Tumor volume was measured over the following days(III; day 0 to day 9 post i.v. injection of bacteria) with calipers. Therelative tumor volume, normalized to the tumor volume at day 0, isindicated (IV) as mm³. The mean is indicated with symbols, error barsdepicted show the standard error of the mean. Statistical significanceis measured with a 2way ANOVA, * indicates p value <0.05, ** a p value<0.005.

FIG. 33 : Tumor progression in wildtype Balb/C mice allografted s.c.with 4T1 breast cancer cells. Wildtype Balb/C mice allografted s.c. with4T1 breast cancer cells were i.v. injected with I: PBS or II: 1*10⁷ Y.enterocolitica dHOPEMT, once the tumor had reached a size of 150-250mm3. The day of the i.v. injection of bacteria was defined as day 0.Tumor volume was measured over the following days (III; day 0 to day 9post i.v. injection of bacteria) with calipers. The relative tumorvolume, normalized to the tumor volume at day 0, is indicated (IV) asmm³. The mean is indicated with symbols, error bars depicted show thestandard error of the mean.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides recombinant Gram-negative bacterialstrains and the use thereof for delivery of repeated domains of aheterologous protein or two or more domains of different heterologousproteins into eukaryotic cells.

For the purposes of interpreting this specification, the followingdefinitions will apply and whenever appropriate, terms used in thesingular will also include the plural and vice versa. It is to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting.

The term “Gram-negative bacterial strain” as used herein includes thefollowing bacteria: Aeromonas salmonicida, Aeromonas hydrophila,Aeromonas veronii, Anaeromyxobacter dehalogenans, Bordetellabronchiseptica, Bordetella parapertussis, Bordetella pertussis,Bradyrhizobium japonicum, Burkholderia cenocepacia, Burkholderiacepacia, Burkholderia mallei, Burkholderia pseudomallei, Chlamydiamuridarum, Chlamydia trachmoatis, Chlamydophila abortus, Chlamydophilapneumoniae, Chromobacterium violaceum, Citrobacter rodentium,Desulfovibrio vulgaris, Edwardsiella tarda, Endozoicomonas elysicola,Erwinia amylovora, Escherichia albertii, Escherichia coli, Lawsoniaintracellularis, Mesorhizobium loti, Myxococcus xanthus, Pantoeaagglomerans, Photobacterium damselae, Photorhabdus luminescens,Photorabdus temperate, Pseudoalteromonas spongiae, Pseudomonasaeruginosa, Pseudomonas plecoglossicida, Pseudomonas syringae, Ralstoniasolanacearum, Rhizobium sp, Salmonella enterica and other Salmonella sp,Shigella flexneri and other Shigella sp, Sodalis glossinidius, Vibrioalginolyticus, Vibrio azureus, Vibrio campellii, Vibrio caribbenthicus,Vibrio harvey, Vibrio parahaemolyticus, Vibrio tasmaniensis, Vibriotubiashii, Xanthomonas axonopodis, Xanthomonas campestris, Xanthomonasoryzae, Yersinia enterocolitica, Yersinia pestis, Yersiniapseudotuberculosis. Preferred Gram-negative bacterial strains of theinvention are Gram-negative bacterial strains comprised by the family ofEnterobacteriaceae and Pseudomonadaceae. The Gram-negative bacterialstrain of the present invention is normally used for delivery ofheterologous proteins by the bacterial T3SS into eukaryotic cells invitro and/or in vivo, preferably in vivo.

The term “recombinant Gram-negative bacterial strain” used herein refersto a Gram-negative bacterial strain genetically transformed with avector. A useful vector of the present invention is e.g an expressionvector, a vector for chromosomal or virulence plasmid insertion or a DNAor RNA fragment for chromosomal or virulence plasmid insertion ormodification.

The term “recombinant Gram-negative bacterial strains which aredeficient in producing at least one T3SS functional effector protein”used herein refers to a recombinant Gram-negative bacterial strain inwhich at least one T3SS effector protein is mutated such that theresulting recombinant Gram-negative bacterial strain no longer producesa functional form of at least one T3SS effector protein i.e. that theexpression of such effector gene is abolished so that the resultingrecombinant Gram-negative bacterial strains does not produce any of theat least one T3SS effector protein or that the catalytic activity of theencoded effector protein is abolished so that the at least one T3SSeffector protein produced does not have its catalytic activity. e.g.does not excersise its effector functions. For the purpose of deliveringproteins, the secretion and translocation system of the recombinantGram-negative bacterial strains which are deficient in producing atleast one T3SS functional effector protein needs to be intact. The term“T3SS effector protein” or “bacterial T3SS effector protein” as usedherein refers to proteins which are naturally injected by T3S systemsinto the cytosol of eukaryotic cells and to proteins which are naturallysecreted by T3S systems that might e.g form the translocation pore intothe eukaryotic membrane (including pore-forming tranlocators (asYersinia YopB and YopD) and tip-proteins like Yersinia LcrV). Preferablyproteins which are naturally injected by T3S systems into the cytosol ofeukaryotic cells are used. These virulence factors will paralyze orreprogram the eukaryotic cell to the benefit of the pathogen. T3Seffectors display a large repertoire of biochemical activities andmodulate the function of crucial host regulatory molecules and includeAvrA, AvrB, AvrBs2, AvrBS3, AvrBsT, AvrD, AvrD1, AvrPphB, AvrPphC,AvrPphEPto, AvrPpiBPto, AvrPto, AvrPtoB, AvrRpm1, AvrRpt2, AvrXv3, CigR,EspF, EspG, EspH, EspZ, ExoS, ExoT, GogB, GtgA, GtgE, GALA family ofproteins, HopAB2, HopAO1, HopI1, HopM1, HopN1, HopPtoD2, HopPtoE,HopPtoF, HopPtoN, HopU1, HsvB, IcsB, IpaA, IpaB, IpaC, IpaH, IpaH7.8,IpaH9.8, IpgB1, IpgB2, IpgD, LcrV, Map, OspC1, OspE2, OspF, OspG, OspI,PipB, PipB2, PopB, PopP2, PthXo1, PthXo6, PthXo7, SifA, SifB, SipA/SspA,SipB, SipC/SspC, SipD/SspD, SlrP, SopA, SopB/SigD, SopD, SopE, SopE2,SpiC/SsaB, SptP, SpvB, SpvC, SrfH, SrfJ, Sse, SseB, SseC, SseD, SseF,SseG, SseI/SrfH, SseJ, SseK1, SseK2, SseK3, SseL, SspH1, SspH2, SteA,SteB, SteC, SteD, SteE, TccP2, Tir, VirA, VirPphA, VopF, XopD, YopB,YopD YopE, YopH, YopJ, YopM, YopO, YopP, YopT, YpkA.

The terms “Gram-negative bacterial strain deficient to produce an aminoacid essential for growth” and “auxotroph mutant” are used hereininterchangeably and refer to Gram-negative bacterial strains which cannot grow in the absence of at least one exogenously provided essentialamino acid or a precursor thereof. The amino acid the strain isdeficient to produce is e.g. aspartate, meso-2,6-diaminopimelic acid,aromatic amino acids or leucine-arginine [8]. Such a strain can begenerated by e.g. deletion of the aspartate-beta-semialdehydedehydrogenase gene (Δasd). Such an auxotroph mutant cannot grow inabsence of exogenous meso-2,6-diaminopimelic acid [9]. The mutation,e.g. deletion of the aspartate-beta-semialdehyde dehydrogenase gene ispreferred herein for a Gram-negative bacterial strain deficient toproduce an amino acid essential for growth of the present invention.

The term “Gram-negative bacterial strain deficient to produce adhesionproteins binding to the eukaryotic cell surface or extracellular matrix”refers to mutant Gram-negative bacterial strains which do not express atleast one adhesion protein compared to the adhesion proteins expressedby the corresponding wild type strain. Adhesion proteins may includee.g. extended polymeric adhesion molecules like pili/fimbriae ornon-fimbrial adhesins. Fimbrial adhesins include type-1 pili (such as E.coli Fim-pili with the FimH adhesin), P-pili (such as Pap-pili with thePapG adhesin from E. coli), type 4 pili (as pilin protein from e.g. P.aeruginosa) or curli (Csg proteins with the CsgA adhesin from S.enterica). Non-fimbrial adhesions include trimeric autotransporteradhesins such as YadA from Y. enterocolitica, BpaA (B. pseudomallei),Hia (H. influenzae), BadA (B. henselae), NadA (N. meningitidis) or UspA1(M. catarrhalis) as well as other autotransporter adhesins such asAIDA-1 (E. coli) as well as other adhesins/invasins such as InvA from Y.enterocolitica or Intimin (E. coli) or members of the Dr-family orAfa-family (E. coli). The terms YadA and InvA as used herein refer toproteins from Y. enterocolitica. The autotransporter YadA [10,11] bindsto different froms of collagen as well as fibronectin, while the invasinInvA [12-14] binds to β-integrins in the eukaryotic cell membrane. Ifthe Gram-negative bacterial strain is a Y. enterocolitica strain thestrain is preferably deficient in InvA and/or YadA.

As used herein, the term “family of Enterobacteriaceae” comprises afamily of gram-negative, rod-shaped, facultatively anaerobic bacteriafound in soil, water, plants, and animals, which frequently occur aspathogens in vertebrates. The bacteria of this family share a similarphysiology and demonstrate a conservation within functional elements andgenes of the respective genomes. As well as being oxidase negative, allmembers of this family are glucose fermenters and most are nitratereducers. Enterobacteriaceae bacteria of the invention may be anybacteria from that family, and specifically includes, but is not limitedto, bacteria of the following genera: Escherichia, Shigella,Edwardsiella, Salmonella, Citrobacter, Klebsiella, Enterobacter,Serratia, Proteus, Erwinia, Morganella, Providencia, or Yersinia. Inmore specific embodiments, the bacterium is of the Escherichia coli,Escherichia blattae, Escherichia fergusonii, Escherichia hermanii,Escherichia vuneris, Salmonella enterica, Salmonella bongori, Shigelladysenteriae, Shigella flexneri, Shigella boydii, Shigella sonnei,Enterobacter aerogenes, Enterobacter gergoviae, Enterobacter sakazakii,Enterobacter cloacae, Enterobacter agglomerans, Klebsiella pneumoniae,Klebsiella oxytoca, Serratia marcescens, Yersinia pseudotuberculosis,Yersinia pestis, Yersinia enterocolitica, Erwinia amylovora, Proteusmirabilis, Proteus vulgaris, Proteus penneri, Proteus hauseri,Providencia alcalifaciens, or Morganella morganii species.

Preferably the Gram-negative bacterial strain is selected from the groupconsisting of the genera Yersinia, Escherichia, Salmonella, Shigella,Pseudomonas, Chlamydia, Erwinia, Pantoea, Vibrio, Burkholderia,Ralstonia, Xanthomonas, Chromobacterium, Sodalis, Citrobacter,Edwardsiella, Rhizobiae, Aeromonas, Photorhabdus, Bordetella andDesulfovibrio, more preferably from the group consisting of the generaYersinia, Escherichia, Salmonella, and Pseudomonas, most preferably fromthe group consisting of the genera Yersinia and Salmonella.

The term “Yersinia” as used herein includes all species of Yersinia,including Yersinia enterocolitica, Yersinia pseudotuberculosis andYersinia pestis. Preferred is Yersinia enterocolitica.

The term “Salmonella” as used herein includes all species of Salmonella,including Salmonella enterica and S. bongori. Preferred is Salmonellaenterica.

“Promoter” as used herein refers to a nucleic acid sequence thatregulates expression of a transcriptional unit. A “promoter region” is aregulatory region capable of binding RNA polymerase in a cell andinitiating transcription of a downstream (3′ direction) coding sequence.Within the promoter region will be found a transcription initiation site(conveniently defined by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase such as the putative −35 region and the Pribnow box. The term“operably linked” when describing the relationship between two DNAregions simply means that they are functionally related to each otherand they are located on the same nucleic acid fragment. A promoter isoperably linked to a structural gene if it controls the transcription ofthe gene and it is located on the same nucleic acid fragment as thegene. Usually the promoter is functional in said Gram-negative bacterialstrain, i.e. the promoter is capable of expressing the fusion protein ofthe present invention, i.e. the promoter is capable of expressing thefusion protein of the present invention without further geneticengineering or expression of further proteins. Furthermore, a functionalpromoter must not be naturally counter-regulated to the bacterial T3SS.

The term “delivery” used herein refers to the transportation of aprotein from a recombinant Gram-negative bacterial strain to aeukaryotic cell, including the steps of expressing the heterologousprotein in the recombinant Gram-negative bacterial strain, secreting theexpressed protein(s) from such Gram-negative bacterial strain andtranslocating the secreted protein(s) by such Gram-negative bacterialstrain into the cytosol of the eukaryotic cell. Accordingly, the terms“delivery signal” or “secretion signal” which are used interchangeablyherein refer to a polypeptide sequence which can be recognized by thesecretion and translocation system of the Gram-negative bacterial strainand directs the delivery of a protein from the Gram-negative bacterialstrain to eukaryotic cells.

The term “delivery signal from a bacterial effector protein” used hereinrefers to a delivery signal from a bacterial effector protein functionalin the recombinant Gram-negative bacterial strain, i.e. which allows anexpressed heterologous protein in the recombinant Gram-negativebacterial strain to be secreted from such recombinant Gram-negativebacterial strain by a secretion system such as e.g. the type IIIsecretion system or to be translocated by such recombinant Gram-negativebacterial strain into the cytosol of a eukaryotic cell by a secretionsystem such as e.g. the type III secretion system. The term “deliverysignal from a bacterial effector protein” used herein also comprises afragment of a delivery signal from a bacterial effector protein i.e.shorter versions of a delivery signal e.g. a delivery signal comprisingup to 10, preferably up to 20, more preferably up to 50, even morepreferably up to 100, in particular up to 140 amino acids of a deliverysignal e.g. of a naturally occurring delivery signal. Thus a nucleotidesequence such as e.g. a DNA sequence encoding a delivery signal from abacterial effector protein may encode a full length delivery signal or afragment thereof wherein the fragment usually comprises usually up to30, preferably up to 60, more preferably up to 150, even more preferablyup to 300, in particular up to 420 nucleic acids.

As used herein, the “secretion” of a protein refers to thetransportation of a heterologous protein outward across the cellmembrane of a recombinant Gram-negative bacterial strain. The“translocation” of a protein refers to the transportation of aheterologous protein from a recombinant Gram-negative bacterial strainacross the plasma membrane of a eukaryotic cell into the cytosol of sucheukaryotic cell.

The term “eukaryotic cells” as used herein includes e.g. the followingeukaryotic cells: Hi-5, HeLa, Hek, HUVECs, 3T3, CHO, Jurkat, Sf-9,HepG2, Vero, MDCK, Mefs, THP-1, J774, RAW, Caco2, NCI60, DU145, Lncap,MCF-7, MDA-MB-438, PC3, T47D, A549, U87, SHSY5Y, Ea.Hy926, Saos-2, 4T1,D2A1, B16F10, and primary human hepatocytes. “Eukaryotic cells” as usedherein, are also referred to as “target cells” or “target eukaryoticcells”.

The term “T3SS effector protein” as used herein refers to proteins whichare naturally injected by T3S systems into the cytosol of eukaryoticcells and to proteins which are naturally secreted by T3S systems thatmight e.g form the translocation pore into the eukaryotic membrane(including pore-forming tranlocators (as Yersinia YopB and YopD) andtip-proteins like Yersinia LcrV). Preferably proteins which arenaturally injected by T3S systems into the cytosol of eukaryotic cellsare used. These virulence factors will paralyze or reprogram theeukaryotic cell to the benefit of the pathogen. T3S effectors display alarge repertoire of biochemical activities and modulate the function ofcrucial host regulatory molecules [2,15] and include AvrA, AvrB, AvrBs2,AvrBS3, AvrBsT, AvrD, AvrD1, AvrPphB, AvrPphC, AvrPphEPto, AvrPpiBPto,AvrPto, AvrPtoB, AvrRpm1, AvrRpt2, AvrXv3, CigR, EspF, EspG, EspH, EspZ,ExoS, ExoT, GogB, GtgA, GtgE, GALA family of proteins, HopAB2, HopAO1,HopI1, HopM1, HopN1, HopPtoD2, HopPtoE, HopPtoF, HopPtoN, HopU1, HsvB,IcsB, IpaA, IpaB, IpaC, IpaH, IpaH7.8, IpaH9.8, IpgB1, IpgB2, IpgD,LcrV, Map, OspC1, OspE2, OspF, OspG, OspI, PipB, PipB2, PopB, PopP2,PthXo1, PthXo6, PthXo7, SifA, SifB, SipA/SspA, SipB, SipC/SspC,SipD/SspD, SlrP, SopA, SopB/SigD, SopD, SopE, SopE2, SpiC/SsaB, SptP,SpvB, SpvC, SrfH, SrfJ, Sse, SseB, SseC, SseD, SseF, SseG, SseI/SrfH,SseJ, SseK1, SseK2, SseK3, SseL, SspH1, SspH2, SteA, SteB, SteC, SteD,SteE, TccP2, Tir, VirA, VirPphA, VopF, XopD, YopB, YopD YopE, YopH,YopJ, YopM, YopO, YopP, YopT, YpkA.

T3SS effector genes of Yersinia have been cloned from e.g. Y.enterocolitica which are YopE, YopH, YopM, YopO, YopP/YopJ, and YopT[16]. The respective effector genes can be cloned from Shigella flexneri(e.g. OspF, IpgD, IpgB1), Salmonella enterica (e.g. SopE, SopB, SptP),P. aeruginosa (e.g ExoS, ExoT, ExoU, ExoY) or E. coli (e.g. Tir, Map,EspF, EspG, EspH, EspZ). The nucleic acid sequences of these genes areavailable to those skilled in the art, e.g., in the Genebank Database(yopH, yopO, yopE, yopP, yopM, yopT from NC 002120 GI:10955536; S.flexneri effector proteins from AF386526.1 GI:18462515; S. entericaeffectors from NC_016810.1 GI:378697983 or FQ312003.1 GI:301156631; P.aeruginosa effectors from AE004091.2 GI:110227054 or CP000438.1GI:115583796 and E. coli effector proteins from NC_011601.1GI:215485161).

For the purpose of the present invention, genes are denoted by lettersof lower case and italicised to be distinguished from proteins. In casethe genes (denoted by letters of lower case and italicised) arefollowing a bacterial species name (like E. coli), they refer to amutation of the corresponding gene in the corresponding bacterialspecies. For example, YopE refers to the effector protein encoded by theyopE gene. Y. enterocolitica yopE represents a Y. enterocolitica havinga mutaion in the yopE gene.

As used herein, the terms “polypeptide”, “peptide”, “protein”,“polypeptidic” and “peptidic” are used interchangeably to designate aseries of amino acid residues connected to each other by peptide bondsbetween the alpha-amino and carboxy groups of adjacent residues.Preferred are proteins which have an amino acid sequence comprising atleast 10 amino acids, more preferably at least 20 amino acids.

According to the present invention, “a domain of a heterologous protein”includes domains of naturally occurring proteins and also includesdomains of artificially engineered proteins. As used herein, the term“domain of a heterologous protein” refers to a domain of a heterologousprotein other than a domain of a T3SS effector protein or a domain otherthan a domain comprising the N-terminal fragment thereof to which it canbe fused to achieve a fusion protein. In particular the domain of aheterologous protein as used herein refers to a domain of a heterologousprotein, which do not belong to the proteome, i.e. the entire naturalprotein complement of the specific recombinant Gram-negative bacterialstrain provided and used by the invention, e.g. which do not belong tothe proteome, i.e. the entire natural protein complement of a specificbacterial strain of the genera Yersinia, Escherichia, Salmonella orPseudomonas. Usually the domain of the heterologous protein is of animalorigin including human origin. Preferably the domain of the heterologousprotein is a domain of a human protein. More preferably the domain ofthe heterologous protein is a domain of a protein selected from thegroup consisting of proteins involved in apoptosis or apoptosisregulation, cell cycle regulators, ankyrin repeat proteins, cellsignaling proteins, reporter proteins, transcription factors, proteases,small GTPases, GPCR related proteins, nanobody fusion constructs andnanobodies, bacterial T3SS effectors, bacterial T4SS effectors and viralproteins. Particular preferably the domain of the heterologous proteinis a domain of a protein selected from the group consisting of proteinsinvolved in apoptosis or apoptosis regulation, cell cycle regulators,ankyrin repeat proteins, reporter proteins, small GTPases, GPCR relatedproteins, nanobody fusion constructs, bacterial T3SS effectors,bacterial T4SS effectors and viral proteins. Even more particularpreferred are domains of heterologous proteins selected from the groupconsisting of proteins involved in apoptosis or apoptosis regulation,cell cycle regulators, and ankyrin repeat proteins. Most preferred aredomains of proteins involved in apoptosis or apoptosis regulation, likeanimal proteins involved in apoptosis or apoptosis regulation,preferably domains of human heterologous proteins involved in apoptosisor apoptosis regulation.

The term “repeated domains of a heterologous protein” as used hereinrefers to a fusion protein consisting of several repetitions of a domainof a heterologous protein, where these domains might either be directlyfused to each other or where a variable linker e.g. a linker between 1and 30, preferably between 2 and 15, more preferably between 3 and 10amino acids might be introduced in between the domains.

Preferably repeated identical domains or repeated domains which have anamino acid sequence identity of more than 80%, usually more than 85%,preferably more than 90%, even more preferably more than 95%, inparticular more than 96%, more particular more than 97%, even moreparticular more than 98%, most particular more than 99% are used. Alsopreferred are identical domains which have an amino acid identity of100%. Preferably two repeated domains, more preferably two repeatedidentical domains or two repeated domains having an amino acid sequenceidentity of more than 90%, preferably more than 95% most preferably 100%are comprised by the fusion protein as referred herein. More than two,e.g. three, four, five or six repeated domains are also contemplated bythe present invention.

The term “two or more domains of different heterologous proteins” asused herein refers to a fusion protein consisting of one or severalrepetitions of at least two domains of different heterologous proteinse.g. at least two domains of heterologous proteins having an amino acidsequence identity of 80% or less, where these different domains mighteither be directly fused to each other or where a variable linker e.g. alinker between 1 and 30, preferably between 2 and 15, more preferablybetween 3 and 10 amino acids might be introduced in between the domains.

Preferably two domains of different heterologous proteins are comprisedby the fusion protein as referred herein. More than two, e.g. three,four, five or six domains of different heterologous proteins are alsocontemplated by the present invention.

The term “heterologous proteins which belong to the same functionalclass of proteins” as used herein refers to heterologous proteins whichhave the same function e.g. heterologous proteins having enzymaticactivity, heterologous proteins which act in the same pathway such ase.g. cell cycle regulation, or share a common specific feature as e.g.belonging to the same class of bacterial effector proteins. Functionalclasses of proteins are e.g. proteins involved in apoptosis or apoptosisregulation, proteins which act as cell cycle regulators, ankyrin repeatproteins, cell signaling proteins, reporter proteins, transcriptionfactors, proteases, small GTPases, GPCR related proteins, nanobodyfusion constructs and nanobodies, bacterial T3SS effectors, bacterialT4SS effectors or viral proteins which act jointly in the biologicalprocess of establishing virulence to eukaryotic cells.

The domain of a heterologous protein expressed by the recombinantGram-negative bacterial strain has usually a molecular weight of between1-50 kDa, preferably between 1-30 kDa, more preferably between 1-20 kDa,most preferably between 1-10 kDa.

According to the present invention “proteins involved in apoptosis orapoptosis regulation” or “human heterologous proteins involved inapoptosis or apoptosis regulation” include, but are not limited to, Bad,Bcl2, Bak, Bmt, Bax, Puma, Noxa, Bim, Bcl-xL, Apaf1, Caspase 9, Caspase3, Caspase 6, Caspase 7, Caspase 10, DFFA, DFFB, ROCK1, APP, CAD, ICAD,CAD, EndoG, AIF, HtrA2, Smac/Diablo, Arts, ATM, ATR, Bok/Mtd, Bmf,Mcl-1(S), IAP family, LC8, PP2B, 14-3-3 proteins, PKA, PKC, PI3K,Erk1/2, p90RSK, TRAF2, TRADD, FADD, Daxx, Caspase8, Caspase2, RIP,RAIDD, MKK7, JNK, FLIPs, FKHR, GSK3, CDKs and their inhibitors like theINK4-family (p16(Ink4a), p15(Ink4b), p18(Ink4c), p19(Ink4d)), and theCip1/Waf1/Kip1-2-family (p21(Cip1/Waf1), p27(Kip1), p57(Kip2).

Preferably Bad, Bmt, Bcl2, Bak, Bax, Puma, Noxa, Bim, Bcl-xL, Caspase9,Caspase3, Caspase6, Caspase7, Smac/Diablo, Bok/Mtd, Bmf, Mcl-1(S), LC8,PP2B, TRADD, Daxx, Caspase8, Caspase2, RIP, RAIDD, FKHR, CDKs and theirinhibitors like the INK4-family (p16(Ink4a), p15(Ink4b), p18(Ink4c),p19(Ink4d)), most preferably BIM, Bid, truncated Bid, FADD, Caspase 3(and subunits thereof), Bax, Bad, Akt, CDKs and their inhibitors likethe INK4-family (p16(Ink4a), p15(Ink4b), p18(Ink4c), p19(Ink4d)) areused [17-19]. Additionally proteins involved in apoptosis or apoptosisregulation include DIVA, Bcl-Xs, Nbk/Bik, Hrk/Dp5, Bid and tBid, Egl-1,Bcl-Gs, Cytochrome C, Beclin, CED-13, BNIP1, BNIP3, Bcl-B, Bcl-W, Ced-9,A1, NR13, Bfl-1, Caspase 1, Caspase 2, Caspase 4, Caspase 5, Caspase 8.

Proteins involved in apoptosis or apoptosis regulation are selected fromthe group consisting of pro-apoptotic proteins, anti-apoptotic proteins,inhibitors of apoptosis-prevention pathways and inhibitors ofpro-survival signalling or pathways. Pro-apoptotic proteins compriseproteins selected form the group consisting of Bax, Bak, Diva, Bcl-Xs,Nbk/Bik, Hrk/Dp5, Bmf, Noxa, Puma, Bim, Bad, Bid and tBid, Bok, Apaf1,Smac/Diablo, BNIP1, BNIP3, Bcl-Gs, Beclin 1, Egl-1 and CED-13,Cytochrome C, FADD, the Caspase family, and CDKs and their inhibitorslike the INK4-family (p16(Ink4a), p15(Ink4b), p18(Ink4c), p19(Ink4d)) orselected from the group consisting of Bax, Bak, Diva, Bcl-Xs, Nbk/Bik,Hrk/Dp5, Bmf, Noxa, Puma, Bim, Bad, Bid and tBid, Bok, Egl-1, Apaf1,Smac/Diablo, BNIP1, BNIP3, Bcl-Gs, Beclin 1, Egl-1 and CED-13,Cytochrome C, FADD, and the Caspase family.

Preferred are Bax, Bak, Diva, Bcl-Xs, Nbk/Bik, Hrk/Dp5, Bmf, Noxa, Puma,Bim, Bad, Bid and tBid, Bok, Egl-1, Apaf1, BNIP1, BNIP3, Bcl-Gs, Beclin1, Egl-1 and CED-13, Smac/Diablo, FADD, the Caspase family, CDKs andtheir inhibitors like the INK4-family (p16(Ink4a), p15(Ink4b),p18(Ink4c), p19(Ink4d)). Equally preferred are Bax, Bak, Diva, Bcl-Xs,Nbk/Bik, Hrk/Dp5, Bmf, Noxa, Puma, Bim, Bad, Bid and tBid, Bok, Apaf1,BNIP1, BNIP3, Bcl-Gs, Beclin 1, Egl-1 and CED-13, Smac/Diablo, FADD, theCaspase family.

Anti-apoptotic proteins comprise proteins selected form the groupconsisting of Bcl-2, Bcl-Xl, Bcl-B, Bcl-W, Mcl-1, Ced-9, A1, NR13, IAPfamily and Bfl-1. Preferred are Bcl-2, Bcl-Xl, Bcl-B, Bcl-W, Mcl-1,Ced-9, A1, NR13 and Bfl-1. Inhibitors of apoptosis-prevention pathwayscomprise proteins selected form the group consisting of Bad, Noxa andCdc25A. Preferred are Bad and Noxa.

Inhibitors of pro-survival signalling or pathways comprise proteinsselected form the group consisting of PTEN, ROCK, PP2A, PHLPP, JNK, p38.Preferred are PTEN, ROCK, PP2A and PHLPP.

In some embodiments the heterologous proteins involved in apoptosis orapoptosis regulation are selected from the group consisting of BH3-onlyproteins, caspases and intracellular signalling proteins of deathreceptor control of apoptosis.

BH3-only proteins comprise proteins selected form the group consistingof Bad, BIM, Bid and tBid, Puma, Bik/Nbk, Bod, Hrk/Dp5, BNIP1, BNIP3,Bmf, Noxa, Mcl-1, Bcl-Gs, Beclin 1, Egl-1 and CED-13. Preferred are Bad,BIM, Bid and tBid. Caspases comprise proteins selected form the groupconsisting of Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5,Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10. Preferred areCaspase 3, Caspase 8 and Caspase 9.

Intracellular signalling proteins of death receptor control of apoptosiscomprise proteins selected form the group consisting of FADD, TRADD,ASC, BAP31, GULP1/CED-6, CIDEA, MFG-E8, CIDEC, RIPK1/RIP1, CRADD,RIPK3/RIP3, Crk, SHB, CrkL, DAXX, the 14-3-3 family, FLIP, DFF40 and 45,PEA-15, SODD. Preferred are FADD and TRADD.

In some embodiments two domains of a heterologous proteins involved inapoptosis or apoptosis regulation are comprised by the Gram-negativebacterial strain and/or the vetcor of the present invention, preferablytwo repeated, more preferably two identical repeated domains of aprotein involved in apoptosis or apoptosis regulation or two domains ofdifferent proteins involved in apoptosis or apoptosis regulation. Insome embodiments two domains of a heterologous proteins involved inapoptosis or apoptosis regulation are comprised by the Gram-negativebacterial strain and/or the vetcor of the present invention, wherein oneis a domain of a pro-apoptotic protein and the other is a domain of aprotein which is an inhibitor of apoptosis-prevention pathways orwherein one is a domain of a a pro-apoptotic protein and the otherdomain is a domain of a protein which is an inhibitor of pro-survivalsignalling or pathways.

Pro-apoptotic proteins encompassed by the present invention have usuallyan alpha helical structure, preferably a hydrophobic helix surrounded byamphipathic helices and usually comprise at least one of BH1, BH2, BH3or BH4 domains, preferably comprise at least one BH3 domain. Usuallypro-apoptotic proteins encompassed by the present invention have noenzymatic activity.

Anti-apoptotic proteins encompassed by the present invention haveusually an alpha helical structure, preferably a hydrophobic helixsurrounded by amphipathic helices and comprises a combination ofdifferent BH1, BH2, BH3 and BH4 domains, preferably a combination ofdifferent BH1, BH2, BH3 and BH4 domains wherein a BH1 and a BH2 domainis present, more preferably BH4-BH3-BH1-BH2, BH1-BH2, BH4-BH1-BH2 orBH3-BH1-BH2 (from N- to the C-terminus). Additionally, proteinscontaining at least one BIR domain are also encompassed.

Inhibitors of apoptosis-prevention pathways encompassed by the presentinvention have usually an alpha helical structure, preferably ahydrophobic helix surrounded by amphipathic helices and usually compriseone BH3 domain.

BH1, BH2, BH3 or BH4 domains are each usually between about 5 to about50 amino acids in length. Thus in some embodiments the domains ofheterologous proteins are selected from the group consisting of domainsof heterologous proteins which are about 5 to about 200, preferablyabout 5 to about 150, more preferably about 5 to about 100, mostpreferably about 5 to about 50, in particular about 5 to about 25 aminoacids in length.

A particular preferred domain is the BH3 domain of apoptosis inducertBID, more particular the BH3 domain comprising a sequence selected fromthe group consisting of SEQ ID NOs: 209, 210, 211 and 212, preferablySEQ ID NO: 211 or SEQ ID NO: 212. Equally preferred is the BH3 domain ofapoptosis regulator BAX, more particular the BAX domain comprising asequence selected from the group consisting of SEQ ID NOs: 213, 214, 215and 216, preferably SEQ ID NO: 215 or SEQ ID NO: 216. The human andmurine sequences are given in SEQ ID NOs 209-216, but tBID and BAX BH3domains of all other species are equally included.

In some embodiments the repeated domains of the heterologous proteinsare the BH3 domain, in particular repeated BH3 domains of apoptosisinducer tBID, more particular two repeated BH3 domains of apoptosisinducer tBID, most particular two repeated BH3 domains of apoptosisinducer tBID comprised by the sequence of SEQ ID NO: 202. Thus in apreferred embodiment the vector of the Gram-negative bacterial strain ofthe present invention comprises a second DNA sequence encoding tworepeated domains of a BH3 domain, more preferably two repeated BH3domains of apoptosis inducer tBID. The two repeated domains arepreferably connected by a linker of 1-30 amino acid length, preferably2-15 amino acids, more preferred 3-10 amino acids long.

In some embodiments the two or more domains of different heterologousproteins are domains of heterologous proteins which belong to the samefunctional class of proteins, preferably the different heterologousproteins of the two or more domains are different heterologous proteinsfrom the class of proteins involved in apoptosis or apoptosisregulation. In a preferred embodiment the two or more domains ofdifferent heterologous proteins are the BH3 domain of apoptosis inducertBID and the BH3 domain of apoptosis regulator BAX, in particular thefused BH3 domains comprised by the sequence of SEQ ID NO: 203. The twodomains of different heterologous proteins are preferably connected by alinker of 1-30 amino acid length, preferably 2-15 amino acids, morepreferred 3-10 amino acids long.

In some embodiments the heterologous proteins is a pro-drug convertingenzyme. In these embodiments the recombinant virulence attenuatedGram-negative bacterial strain expresses, preferably expresses andsecretes a pro-drug converting enzyme. A prodrug converting enzyme asreferred herein comprises enzymes converting non-toxic prodrugs into atoxic drug, preferably enzymes seleted from the group consisting ofcytosine deaminase, purine nucleoside phosphorylase, thymidine kinase,beta-galactosidase, carboxylesterases, nitroreductase, carboxypeptidasesand beta-glucuronidases. more preferably enzymes seleted from the groupconsisting of cytosine deaminase, purine nucleoside phosphorylase,thymidine kinase, and beta-galactosidase.

The term “protease cleavage site” as used herein refers to a specificamino acid motif within an amino acid sequence e.g. within an amino acidsequence of a protein or a fusion protein, which is cleaved by aspecific protease, which recognizes the amino acid motif. For review see[20]. Examples of protease cleavage sites are amino acid motifs, whichare cleaved by a protease selected from the group consisting ofenterokinase (light chain), enteropeptidase, prescission protease, humanrhinovirus protease (HRV 3C), TEV protease, TVMV protease, FactorXaprotease and thrombin. The following amino acid motif is recognized bythe respective protease:

-   -   Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 226): Enterokinase (light        chain)/Enteropeptidase    -   Leu-Glu-Val-Leu-Phe-Gln/Gly-Pro (SEQ ID NO: 227): PreScission        Protease/human Rhinovirus protease (HRV 3C)    -   Glu-Asn-Leu-Tyr-Phe-Gln-Ser (SEQ ID NO:228) and modified motifs        based on the Glu-X-X-Tyr-X-Gln-Gly/Ser (SEQ ID NO: 229) (where X        is any amino acid) recognized by TEV protease (tobacco etch        virus)    -   Glu-Thr-Val-Arg-Phe-Gln-Ser (SEQ ID NO: 230): TVMV protease    -   Ile-(Glu or Asp)-Gly-Arg (SEQ ID NO: 231): FactorXa protease    -   Leu-Val-Pro-Arg/Gly-Ser (SEQ ID NO: 232): Thrombin.

Encompassed by the protease cleavage sites as used herein is ubiquitin.Thus in some preferred embodiments ubiquitin is used as proteasecleavage site, i.e. the third DNA sequence encodes ubiquitin as proteasecleavage site, which can be cleaved by a specific ubiquitin processingproteases at the N-terminal site, e.g. which can be cleaved by aspecific ubiquitin processing proteases called Deubiquitinating enzymesat the N-terminal site endogeneously in the cell where the fusionprotein has been delivered to. Ubiquitin is processed at its C-terminusby a group of endogenous Ubiquitin-specific C-terminal proteases(Deubiquitinating enzymes, DUBs). The cleavage of Ubiquitin by DUBs issupposed to happen at the very C-terminus of Ubiquitin (after G76).

An “individual,” “subject” or “patient” is a vertebrate. In certainembodiments, the vertebrate is a mammal. Mammals include, but are notlimited to, primates (including human and non-human primates) androdents (e.g., mice and rats). In certain embodiments, a mammal is ahuman.

The term “mutation” is used herein as a general term and includeschanges of both single base pair and multiple base pairs. Such mutationsmay include substitutions, frame-shift mutations, deletions, insertionsand truncations.

The term “labelling molecule or an acceptor site for a labellingmolecule” as used herein refers to a small chemical compound binding toa specific amino acid sequence resulting in fluorescence of the boundchemical compound, preferably coumarine ligase/coumarine acceptor site(and derivates thereof), resorufin ligase/resorufin acceptor site (andderivates thereof) and the tetra-Cysteine motif (asCys-Cys-Pro-Gly-Cys-Cys (SEQ ID NO: 233) and derivates thereof) in usewith FlAsH/ReAsH dye (life technologies) or a fluorescent protein asEnhanced Green Fluorescent Protein (EGFP).

The term “nuclear localization signal” as used herein refers to an aminoacid sequence that marks a protein for import into the nucleus of aeukaryotic cell and includes preferably a viral nuclear localizationsignal such as the SV40 large T-antigen derived NLS (PPKKKRKV) (SEQ IDNO: 234).

The term “multiple cloning site” as used herein refers to a short DNAsequence containing several restriction sites for cleavage byrestriction endonucleases such as AclI, HindIII, SspI, MluCI, Tsp509I,PciI, AgeI, BspMI, BfuAI, SexAI, MluI, BceAI, HpyCH4IV, HpyCH4III, BaeI,BsaXI, AflIII, SpeI, BsrI, BmrI, BglII, AfeI, AluI, StuI, ScaI, ClaI,BspDI, PI-SceI, NsiI, AseI, SwaI, CspCI, MfeI, BssSI, BmgBI, PmlI,DraIII, AleI, EcoP15I, PvuII, AlwNI, BtsIMutI, TspRI, NdeI, NlaIII,CviAII, FatI, MslI, FspEI, XcmI, BstXI, PflMI, BccI, NcoI, BseYI, FauI,SmaI, XmaI, TspMI, Nt.CviPII, LpnPI, AciI, SacII, BsrBI, MspI, HpaII,ScrFI, BssKI, StyD4I, BsaJI, BslI, BtgI, NciI, AvrII, MnlI, BbvCI,Nb.BbvCI, Nt.BbvCI, SbfI, Bpu10I, Bsu36I, EcoNI, HpyAV, BstNI, PspGI,StyI, BcgI, PvuI, BstUI, EagI, RsrII, BsiEI, BsiWI, BsmBI, Hpy99I,MspA1I, MspJI, SgrAI, BfaI, BspCNI, XhoI, EarI, AcuI, PstI, BpmI, DdeI,SfcI, AflII, BpuEI, SmlI, AvaI, BsoBI, MboII, BbsI, XmnI, BsmI, Nb.BsmI,EcoRI, HgaI, AatII, ZraI, Tth111I PflFI, PshAI, AhdI, DrdI, Eco53kI,SacI, BseRI, PleI, Nt.BstNBI, MlyI, HinfI, EcoRV, MboI, Sau3AI, DpnIIBfuCI, DpnI, BsaBI, TfiI, BsrDI, Nb.BsrDI, BbvI, BtsI, Nb.BtsI, BstAPI,SfaNI, SphI, NmeAIII, NaeI, NgoMIV, BglI, AsiSI, BtgZI, HinP1I, HhaI,BssHII, NotI, Fnu4HI, Cac8I, MwoI, NheI, BmtI, SapI, BspQI, Nt.BspQI,BlpI, TseI, ApeKI, Bsp1286I, AlwI, Nt.AlwI, BamHI, FokI, BtsCI, HaeIII,PhoI, FseI, SfiI, NarI, KasI, SfoI, PluTI, AscI, EciI, BsmFI, ApaI,PspOMI, Sau96I, NlaIV, KpnI, Acc65I, BsaI, HphI, BstEII, AvaII, BanI,BaeGI, BsaHI, BanII, RsaI, CviQI, BstZ17I, BciVI, SalI, Nt.BsmAI, BsmAI,BcoDI, ApaLI, BsgI, AccI, Hpy166II, Tsp45I, HpaI, PmeI, HincII, BsiHKAI,ApoI, NspI, BsrFI, BstYI, HaeII, CviKI-1, EcoO109I, PpuMI, I-CeuI,SnaBI, I-SceI, BspHI, BspEI, MmeI, TaqαI, NruI, Hpy188I, Hpy188III,XbaI, BclI, HpyCH4V, FspI, PI-PspI, MscI, BsrGI, MseI, Pad, PsiI, BstBI,DraI, PspXI, BsaWI, BsaAI, EaeI, preferably XhoI, XbaI, HindIII, NcoI,NotI, EcoRI, EcoRV, BamHI, NheI, SacI, SalI, BstBI. The term “multiplecloning site” as used herein further refers to a short DNA sequence usedfor recombination events as e.g in Gateway cloning strategy or formethods such as Gibbson assembly or topo cloning.

The term “Yersinia wild type strain” as used herein refers to anaturally occurring variant (as Y. enterocolitica E40) or a naturallyoccurring variant containing genetic modifications allowing the use ofvectors, such as deletion mutations in restriction endonucleases orantibiotic resistance genes (as Y. enterocolitica MRS40, the Ampicillinsensitive derivate of Y. enterocolitica E40) These strains containchromosomal DNA as well as an unmodified virulence plasmid (called pYV).

The term “comprise” is generally used in the sense of include, that isto say permitting the presence of one or more features or components.

The term “about” refers to a range of values±10% of a specified value.For example, the phrase “about 200” includes ±10% of 200, or from 180 to220.

In one embodiment of the present invention the recombinant Gram-negativebacterial strain is transformed with a vector which comprises in the 5′to 3′ direction:

a first DNA sequence encoding a delivery signal or a fragment thereoffrom a bacterial effector protein;

a second DNA sequence encoding repeated domains of a heterologousprotein or two or more domains of different heterologous proteins fusedin frame to the 3′ end of said first DNA sequence, wherein theheterologous proteins are selected from the group consisting of proteinsinvolved in apoptosis or apoptosis regulation, cell cycle regulators,ankyrin repeat proteins, cell signaling proteins, reporter proteins,transcription factors, proteases, small GTPases, GPCR related proteins,nanobody fusion constructs and nanobodies, bacterial T3SS effectors,bacterial T4SS effectors and viral proteins. Preferably the DNA sequenceencoding repeated domains of a heterologous protein or two or moredomains of different heterologous proteins is flanked on its 3′ end by aDNA sequence homologous to the DNA sequence of the chromosome or of theendogenous virulence plasmid at the 3′ end of the delivery signal from abacterial effector protein or to a fragment thereof. More preferably,this DNA sequence flanking the homologous protein on its 3′ end ishomologous to the DNA sequence and is lying within 10 kbp on thechromosome or on an endogenous virulence plasmid at the 3′ end of thedelivery signal from a bacterial effector protein or to a fragmentthereof. In particular, this nucleotide sequence flanking the homologousprotein on its 3′ end is homologous to the DNA sequence and is withinthe same operon on the chromosome or on an endogenous virulence plasmidas the delivery signal from a bacterial effector protein or a fragmentthereof. In this embodiment, transformation is usually performed so thatthe fused first and the second DNA sequence are inserted by homologousrecombination on an endogenous virulence plasmid or a chromosome,preferably on an endogenous virulence plasmid, of the recombinantvirulence attenuated Gram-negative bacterial strain, and the fused firstand the second DNA sequence is operably linked to a promoter of anendogenous virulence plasmid or of a chromosome e.g. of a chromosomalpathogenicity island. Preferably the fused first and the second DNAsequence is operably linked to a promoter of an endogenous virulenceplasmid. In this embodiment the first DNA sequence comprises a deliverysignal or fragment thereof from a bacterial effector protein, preferablya fragment thereof, which provides for homologous recombination at thehomologous site at the chromosome or at an endogenous virulence plasmidto result in the second DNA sequence be placed in frame to the 3′ end ofthe chromosomal or endogenous virulence plasmid delivery signal which isoperatively linked to the endogenous promoter.

In a further embodiment of the present invention the recombinantGram-negative bacterial strain, is transformed with a nucleotidemolecule, preferably a DNA nucleotide molecule, comprising a nucleotidesequence encoding repeated domains of a heterologous protein or two ormore domains of different heterologous proteins and a nucleotidesequence which is homologous or identical to a nucleotide sequenceencoding a delivery signal from a bacterial effector protein or which ishomologous or identical to a nucleotide sequence encoding a fragment ofa delivery signal from a bacterial effector protein, wherein thedelivery signal from a bacterial effector protein is encoded on thechromosome or on an endogenous virulence plasmid of the recombinantvirulence attenuated Gram-negative bacterial strain. Preferably thenucleotide sequence which is homologous or identical to a nucleotidesequence of a delivery signal from a bacterial effector protein or to afragment thereof is located on the 5′ end of the nucleotide sequenceencoding repeated domains of a heterologous protein or two or moredomains of different heterologous proteins. More preferably thenucleotide sequence encoding repeated domains of a heterologous proteinor two or more domains of different heterologous proteins is flanked onits 3′ end by a nucleotide sequence homologous to the DNA sequence ofthe chromosome or of the endogenous virulence plasmid at the 3′ end ofthe delivery signal from a bacterial effector protein or to a fragmentthereof. Even more preferably, this nucleotide sequence flanking thehomologous protein on its 3′ end is homologous to the DNA sequence lyingwithin 10 kbp on the chromosome or on an endogenous virulence plasmid atthe 3′ end of the delivery signal from a bacterial effector protein orto a fragment thereof. In particular, this nucleotide sequence flankingthe homologous protein on its 3′ end is homologous to the DNA sequenceis within the same operon on the chromosome or on an endogenousvirulence plasmid as the delivery signal from a bacterial effectorprotein or a fragment thereof. In this embodiment, transformation isusually performed so that the nucleotide sequence encoding repeateddomains of a heterologous protein or two or more domains of differentheterologous proteins is inserted on an endogenous virulence plasmid ora chromosome of the recombinant virulence attenuated Gram-negativebacterial strain at the 3′ end of a delivery signal from a bacterialeffector protein encoded by the chromosome or the endogenous virulenceplasmid, wherein the repeated domains of a heterologous protein or twoor more domains of different heterologous proteins fused to the deliverysignal are expressed and secreted.

In case the recombinant virulence attenuated Gram-negative bacterialstrain is a Yersinia strain the endogenous virulence plasmid forinsertion is pYV (plasmid of Yersinia Virulence). In case therecombinant virulence attenuated Gram-negative bacterial strain is aSalmonella strain, the endogenous location for insertion is one of thegene clusters called SpiI or SpiII (for Salmonella pathogenicityisland), a position where an effector protein is elsewhere encoded oralternatively one of the Salmonella virulence plasmids (SVPs).

Preferably the first and the second DNA sequence or the nucleotidemolecule are inserted on an endogenous virulence plasmid at the nativesite of a bacterial effector protein e.g. at the native site of avirulence factor, preferably in case the recombinant virulenceattenuated Gram-negative bacterial strain is a Yersinia strain, at thenative site of YopE or another Yop (YopH, YopO, YopP, YopM, YopT),preferably at the native site of YopE or in case the recombinantvirulence attenuated Gram-negative bacterial strain is a Salmonellastrain at the native site of an effector protein encoded within SpiI,SpiII or encoded elsewhere, preferably at the native site of an effectorprotein encoded within SpiI or SpiII, more preferably at the native siteof SopE or SteA. Preferably the first and the second DNA sequence or thenucleotide molecule are operably linked to a native promoter of abacterial effector protein present on an endogenous virulence plasmide.g. in case the recombinant virulence attenuated Gram-negativebacterial strain is a Yersinia strain to a native promoter from aYersinia virulon gene as outlined below, more preferably to the nativeYopE promoter or another Yop (YopH, YopO, YopP, YopM, YopT) promoter,preferably to the native YopE promoter or in case the recombinantvirulence attenuated Gram-negative bacterial strain is a Salmonellastrain to a native promoter from SpiI or SpiII pathogenicity island orfrom an effector protein elsewhere encoded as outlined below, morepreferably to the native SopE, InvB or SteA promoter.

In one embodiment the present invention provides a recombinantGram-negative bacterial strain, wherein the Gram-negative bacterialstrain is selected from the group consisting of the genera Yersinia,Escherichia, Salmonella and Pseudomonas. In one embodiment the presentinvention provides a recombinant Gram-negative bacterial strain, whereinthe Gram-negative bacterial strain is selected from the group consistingof the genera Yersinia and Salmonella. Preferably the Gram-negativebacterial strain is a Yersinia strain, more preferably a Yersiniaenterocolitica strain. Most preferred is Yersinia enterocolitica E40(0:9, biotype 2) [21] or Ampicilline sensitive derivates thereof as Y.enterocolitica MRS40 (also named Y. enterocolitica subsp. palearcticaMRS40) as described in [22]. Y. enterocolitica E40 and its derivate Y.enterocolitica MRS40 as described in [22] is identical to Y.enterocolitica subsp. palearctica E40 and its derivate Y. enterocoliticasubsp. palearctica MRS40 as described in [23-25]. Also preferably theGram-negative bacterial strain is a Salmonella strain, more preferably aSalmonella enterica strain. Most preferred is Salmonella entericaSerovar Typhimurium SL1344 as described by the Public health Englandculture collection (NCTC 13347).

In one embodiment of the present invention the delivery signal from abacterial T3SS effector protein comprises a bacterial T3SS effectorprotein or a N-terminal fragment thereof wherein the T3SS effectorprotein or a N-terminal fragment thereof may comprise a chaperonebinding site. A T3SS effector protein or a N-terminal fragment thereofwhich comprises a chaperone binding site is particular useful asdelivery signal in the present invention. Preferred T3SS effectorproteins or N-terminal fragments thereof are selected from the groupconsisting of SopE, SopE2, SptP, YopE, ExoS, SipA, SipB, SipD, SopA,SopB, SopD, IpgB1, IpgD, SipC, SifA, SseJ, Sse, SrfH, YopJ, AvrA,AvrBsT, YopT, YopH, YpkA, Tir, EspF, TccP2, IpgB2, OspF, Map, OspG,OspI, IpaH, SspH1, VopF, ExoS, ExoT, HopAB2, XopD, AvrRpt2, HopAO1,HopPtoD2, HopU1, GALA family of proteins, AvrBs2, AvrD1, AvrBS3, YopO,YopP, YopE, YopM, YopT, EspG, EspH, EspZ, IpaA, IpaB, IpaC, VirA, IcsB,OspC1, OspE2, IpaH9.8, IpaH7.8, AvrB, AvrD, AvrPphB, AvrPphC,AvrPphEPto, AvrPpiBPto, AvrPto, AvrPtoB, VirPphA, AvrRpm1, HopPtoE,HopPtoF, HopPtoN, PopB, PopP2, AvrBs3, XopD, and AvrXv3. More preferredT3SS effector proteins or N-terminal fragments thereof are selected fromthe group consisting of SopE, SptP, YopE, ExoS, SopB, IpgB1, IpgD, YopJ,YopH, EspF, OspF, ExoS, YopO, YopP, YopE, YopM, YopT, whereof mostpreferred T3SS effector proteins or N-terminal fragments thereof areselected from the group consisting of IpgB1, SopE, SopB, SptP, OspF,IpgD, YopH, YopO, YopP, YopE, YopM, YopT, in particular YopE or anN-terminal fragment thereof.

Equally preferred T3SS effector proteins or N-terminal fragments thereofare selected from the group consisting of SopE, SopE2, SptP, SteA, SipA,SipB, SipD, SopA, SopB, SopD, IpgB1, IpgD, SipC, SifA, SifB, SseJ, Sse,SrfH, YopJ, AvrA, AvrBsT, YopH, YpkA, Tir, EspF, TccP2, IpgB2, OspF,Map, OspG, OspI, IpaH, VopF, ExoS, ExoT, HopAB2, AvrRpt2, HopAO1, HopU1,GALA family of proteins, AvrBs2, AvrD1, YopO, YopP, YopE, YopT, EspG,EspH, EspZ, IpaA, IpaB, IpaC, VirA, IcsB, OspC1, OspE2, IpaH9.8,IpaH7.8, AvrB, AvrD, AvrPphB, AvrPphC, AvrPphEPto, AvrPpiBPto, AvrPto,AvrPtoB, VirPphA, AvrRpm1, HopPtoD2, HopPtoE, HopPtoF, HopPtoN, PopB,PopP2, AvrBs3, XopD, and AvrXv3. Equally more preferred T3SS effectorproteins or N-terminal fragments thereof are selected from the groupconsisting of SopE, SptP, SteA, SifB, SopB, IpgB1, IpgD, YopJ, YopH,EspF, OspF, ExoS, YopO, YopP, YopE, YopT, whereof equally most preferredT3SS effector proteins or N-terminal fragments thereof are selected fromthe group consisting of IpgB1, SopE, SopB, SptP, SteA, SifB, OspF, IpgD,YopH, YopO, YopP, YopE, and YopT, in particular SopE, SteA, or YopE oran N-terminal fragment thereof, more particular SteA or YopE or anN-terminal fragment thereof, most particular YopE or an N-terminalfragment thereof.

In some embodiments the delivery signal from the bacterial T3SS effectorprotein encoded by the first DNA sequence comprises the bacterial T3SSeffector protein or an N-terminal fragment thereof, wherein theN-terminal fragment thereof includes at least the first 10, preferablyat least the first 20, more preferably at least the first 100 aminoacids of the bacterial T3SS effector protein.

In some embodiments the delivery signal from the bacterial T3SS effectorprotein encoded by the first DNA sequence comprises the bacterial T3SSeffector protein or an N-terminal fragment thereof, wherein thebacterial T3SS effector protein or the N-terminal fragment thereofcomprises a chaperone binding site.

Preferred T3SS effector proteins or a N-terminal fragment thereof, whichcomprise a chaperone binding site comprise the following combinations ofchaperone binding site and T3SS effector protein or N-terminal fragmentthereof: SycE-YopE, InvB-SopE, SicP-SptP, SycT-YopT, SycO-YopO,SycN/YscB-YopN, SycH-YopH, SpcS-ExoS, CesF-EspF, SycD-YopB, SycD-YopD.More preferred are SycE-YopE, InvB-SopE, SycT-YopT, SycO-YopO,SycN/YscB-YopN, SycH-YopH, SpcS-ExoS, CesF-EspF. Most preferred is aYopE or an N-terminal fragment thereof comprising the SycE chaperonebinding site such as an N-terminal fragment of a YopE effector proteincontaining the N-terminal 138 amino acids of the YopE effector proteindesignated herein as YopE₁₋₁₃₈ and as shown in SEQ ID NO. 2 or a SopE oran N-terminal fragment thereof comprising the InvB chaperone bindingsite s as uch an N-terminal fragment of a SopE effector proteincontaining the N-terminal 81 or 105 amino acids of the SopE effectorprotein designated herein as SopE₁₋₈₁ or SopE₁₋₁₀₅ respectively, and asshown in SEQ ID NO.: 142 or 143.

In one embodiment of the present invention the recombinant Gram-negativebacterial strain is a Yersinia strain and the delivery signal from thebacterial T3SS effector protein encoded by the first DNA sequencecomprises a YopE effector protein or an N-terminal part, preferably theY. enterocolitica YopE effector protein or an N-terminal part thereof.Preferably the SycE binding site is comprised within the N-terminal partof the YopE effector protein. In this connection an N-terminal fragmentof a YopE effector protein may comprise the N-terminal 12, 16, 18, 52,53, 80 or 138 amino acids [26-28]. Most preferred is an N-terminalfragment of a YopE effector protein containing the N-terminal 138 aminoacids of the YopE effector protein e.g. as described in Forsberg andWolf-Watz [29] designated herein as YopE₁₋₁₃₈ and as shown in SEQ IDNO.: 2.

In one embodiment of the present invention the recombinant Gram-negativebacterial strain is a Salmonella strain and the delivery signal from thebacterial T3SS effector protein encoded by the first DNA sequencecomprises a SopE or SteA effector protein or an N-terminal part thereof,preferably the Salmonella enterica SopE or SteA effector protein or anN-terminal part thereof. Preferably the chaperon binding site iscomprised within the N-terminal part of the SopE effector protein. Inthis connection an N-terminal fragment of a SopE effector proteinprotein may comprise the N-terminal 81 or 105 amino acids. Mostpreferred is the full length SteA and an N-terminal fragment of a SopEeffector protein containing the N-terminal 105 amino acids of theeffector protein e.g. as described in SEQ ID NO. 142 or 143.

One skilled in the art is familiar with methods for identifying thepolypeptide sequences of an effector protein that are capable ofdelivering a protein. For example, one such method is described by Soryet al. [21]. Briefly, polypeptide sequences from e.g. various portionsof the Yop proteins can be fused in-frame to a reporter enzyme such asthe calmodulin-activated adenylate cyclase domain (or Cya) of theBordetella pertussis cyclolysin. Delivery of a Yop-Cya hybrid proteininto the cytosol of eukaryotic cells is indicated by the appearance ofcyclase activity in the infected eukaryotic cells that leads to theaccumulation of cAMP. By employing such an approach, one skilled in theart can determine, if desired, the minimal sequence requirement, i.e., acontiguous amino acid sequence of the shortest length, that is capableof delivering a protein, see, e.g. [21]. Accordingly, preferred deliverysignals of the present invention consists of at least the minimalsequence of amino acids of a T3SS effector protein that is capable ofdelivering a protein.

In one embodiment the present invention provides mutant recombinantGram-negative bacterial strains in particular recombinant Gram-negativebacterial strains which are deficient in producing at least one T3SSfunctional effector protein.

According to the present invention, such a mutant Gram-negativebacterial strain e.g. such a mutant Yersinia strain can be generated byintroducing at least one mutation into at least one effector-encodinggene. Preferably, such effector-encoding genes include YopE, YopH,YopO/YpkA, YopM, YopP/YopJ and YopT as far as a Yersinia strain isconcerned. Preferably, such effector-encoding genes include AvrA, CigR,GogB, GtgA, GtgE, PipB, SifB, SipA/SspA, SipB, SipC/SspC, SipD/SspD,SlrP, SopB/SigD, SopA, SpiC/SsaB, SseB, SseC, SseD, SseF, SseG,SseI/SrfH, SopD, SopE, SopE2, SspH1, SspH2, PipB2, SifA, SopD2, SseJ,SseK1, SseK2, SseK3, SseL, SteC, SteA, SteB, SteD, SteE, SpvB, SpvC,SpvD, SrfJ, SptP, as far as a Salmonella strain is concerned. Mostpreferably, all effector-encoding genes are deleted. The skilled artisanmay employ any number of standard techniques to generate mutations inthese T3SS effector genes. Sambrook et al. describe in general suchtechniques. See Sambrook et al. [30].

In accordance with the present invention, the mutation can be generatedin the promoter region of an effector-encoding gene so that theexpression of such effector gene is abolished.

The mutation can also be generated in the coding region of aneffector-encoding gene such that the catalytic activity of the encodedeffector protein is abolished. The “catalytic activity” of an effectorprotein refers normally to the anti-target cell function of an effectorprotein, i.e., toxicity. Such activity is governed by the catalyticmotifs in the catalytic domain of an effector protein. The approachesfor identifying the catalytic domain and/or the catalytic motifs of aneffector protein are well known by those skilled in the art. See, forexample, [31,32].

Accordingly, one preferred mutation of the present invention is adeletion of the entire catalytic domain. Another preferred mutation is aframeshift mutation in an effector-encoding gene such that the catalyticdomain is not present in the protein product expressed from such“frameshifted” gene. A most preferred mutation is a mutation with thedeletion of the entire coding region of the effector protein. Othermutations are also contemplated by the present invention, such as smalldeletions or base pair substitutions, which are generated in thecatalytic motifs of an effector protein leading to destruction of thecatalytic activity of a given effector protein.

The mutations that are generated in the genes of the T3SS functionaleffector proteins may be introduced into the particular strain by anumber of methods. One such method involves cloning a mutated gene intoa “suicide” vector which is capable of introducing the mutated sequenceinto the strain via allelic exchange. An example of such a “suicide”vector is described by [33].

In this manner, mutations generated in multiple genes may be introducedsuccessively into a Gram-negative bacterial strain giving rise topolymutant, e.g a sixtuple mutant recombinant strain. The order in whichthese mutated sequences are introduced is not important. Under somecircumstances, it may be desired to mutate only some but not all of theeffector genes. Accordingly, the present invention further contemplatespolymutant Yersinia other than sixtuple-mutant Yersinia, e.g.,double-mutant, triple-mutant, quadruple-mutant and quintuple-mutantstrains. For the purpose of delivering proteins, the secretion andtranslocation system of the instant mutant strain needs to be intact.

A preferred recombinant Gram-negative bacterial strain of the presentinvention is a recombinant Gram-negative bacterial strain which isdeficient in producing at least one, preferably at least two, morepreferably at least three, even more preferably at least four, inparticular at least five, more particular at least six, most particularall T3SS effector proteins e.g. a sixtuple-mutant Gram-negativebacterial strain in which all the effector-encoding genes are mutatedsuch that the resulting Gram-negative bacterial strain no longer produceany functional effector proteins.

A more preferred recombinant Gram-negative bacterial strain of thepresent invention is a sixtuple-mutant Yersinia strain in which all theeffector-encoding genes are mutated such that the resulting Yersinia nolonger produce any functional effector proteins. Such sixtuple-mutantYersinia strain is designated as ΔyopH,O,P,E,M,T for Y. enterocolitica.As an example such a sixtuple-mutant can be produced from the Y.enterocolitica MRS40 strain giving rise to Y. enterocolitica MRS40ΔyopH,O,P,E,M,T, which is preferred.

A further aspect of the present invention is directed to a vector foruse in combination with the recombinant Gram-negative bacterial strainsto deliver a desired protein into eukaryotic cells, wherein the vectorcomprises in the 5′ to 3′ direction:

a promoter;

a first DNA sequence encoding a delivery signal from a bacterial T3SSeffector protein, operably linked to said promoter;

a second DNA sequence encoding repeated domains of a heterologousprotein or two or more domains of different heterologous proteins fusedin frame to the 3′ end of said first DNA sequence,

wherein the heterologous proteins are selected from the group consistingof proteins involved in apoptosis or apoptosis regulation, cell cycleregulators, ankyrin repeat proteins, cell signaling proteins, reporterproteins, transcription factors, proteases, small GTPases, GPCR relatedproteins, nanobody fusion constructs and nanobodies, bacterial T3SSeffectors, bacterial T4SS effectors and viral proteins.

Promoter, heterologous protein and protease cleavage site as describedsupra can be used for the vector of the Gram-negative bacterial strain.

A further aspect of the present invention is directed to a vector foruse in combination with the recombinant Gram-negative bacterial strainsto deliver a desired protein into eukaryotic cells, wherein the vectorcomprises in the 5′ to 3′ direction:

a first DNA sequence encoding a delivery signal or a fragment thereoffrom a bacterial effector protein;

a second DNA sequence encoding repeated domains of a heterologousprotein or two or more domains of different heterologous proteins fusedin frame to the 3′ end of said first DNA sequence,

wherein the heterologous proteins are selected from the group consistingof proteins involved in apoptosis or apoptosis regulation, cell cycleregulators, ankyrin repeat proteins, cell signaling proteins, reporterproteins, transcription factors, proteases, small GTPases, GPCR relatedproteins, nanobody fusion constructs and nanobodies, bacterial T3SSeffectors, bacterial T4SS effectors and viral proteins.

Preferably the DNA sequence encoding repeated domains of a heterologousprotein or two or more domains of different heterologous proteins of thevector is flanked on its 3′ end by a DNA sequence homologous to the DNAsequence of the chromosome or of the endogenous virulence plasmid at the3′ end of the delivery signal from a bacterial effector protein or to afragment thereof. More preferably, this DNA sequence flanking thehomologous protein on its 3′ end is homologous to the DNA sequence andis lying within 10 kbp on the chromosome or on an endogenous virulenceplasmid at the 3′ end of the delivery signal from a bacterial effectorprotein or to a fragment thereof. In particular, this nucleotidesequence flanking the homologous protein on its 3′ end is homologous tothe DNA sequence and is within the same operon on the chromosome or onan endogenous virulence plasmid as the delivery signal from a bacterialeffector protein or a fragment thereof. Heterologous protein andprotease cleavage site as described supra can be used for the vector ofthe Gram-negative bacterial strain.

Vectors which can be used according to the invention depend on theGram-negative bacterial strains used as known to the skilled person.Vectors which can be used according to the invention include expressionvectors (including synthetic or otherwise generated modified versions ofendogenous virulence plasmids), vectors for chromosomal or virulenceplasmid insertion and DNA fragments for chromosomal or virulence plasmidinsertion. Expression vectors which are useful in e.g. Yersinia,Escherichia, Salmonella or Pseudomonas strain are e.g pUC, pBad, pACYC,pUCP20 and pET plasmids. Vectors for chromosomal or virulence plasmidinsertion which are useful in e.g. Yersinia, Escherichia, Salmonella orPseudomonas strain are e.g pKNG101. DNA fragments for chromosomal orvirulence plasmid insertion refer to methods used in e.g. Yersinia,Escherichia, Salmonella or Pseudomonas strain as e.g. lambda-red geneticengineering. Vectors for chromosomal or virulence plasmid insertion orDNA fragments for chromosomal or virulence plasmid insertion may insertthe first, second and/or third DNA sequence of the present invention sothat the first, second and/or third DNA sequence is operably linked toan endogenous promoter of the recombinant Gram-negative bacterialstrain. Thus if a vector for chromosomal or virulence plasmid insertionor a DNA fragment for chromosomal or virulence plasmid insertion isused, an endogenous promoter can be encoded on the endogenous bacterialDNA (chromosomal or plasmid DNA) and only the first and second DNAsequence will be provided by the engineered vector for chromosomal orvirulence plasmid insertion or DNA fragment for chromosomal or virulenceplasmid insertion. Alternatively, if a vector for chromosomal orvirulence plasmid insertion or a nucleotide molecule such as e.g. a DNAsequence for chromosomal or virulence plasmid insertion is used, anendogenous promoter and the delivery signal from a bacterial effectorprotein can be encoded on the endogenous bacterial DNA (chromosomal orplasmid DNA) and only the nucleotide molecule such as e.g. a DNAsequence encoding the heterologous protein will be provided by a vectorfor chromosomal or virulence plasmid insertion or by a nucleotidemolecule such as e.g. a DNA sequence for chromosomal or virulenceplasmid insertion. Thus a promoter is not necessarily needed to becomprised by the vector used for transformation of the recombinantGram-negative bacterial strains i.e. the recombinant Gram-negativebacterial strains of the present invention may be transformed with avector which dose not comprise a promoter. The vector of the presentinvention is normally used for delivery of the heterologous proteins bythe bacterial T3SS into eukaryotic cells in vitro and in vivo.

A preferred vector e.g. a preferred expression vector for Yersinia isselected from the group consisting of pBad_Si1 and pBad_Si2. pBad_Si2was constructed by cloning of the SycE-YopE₁₋₁₃₈ fragment containingendogenous promoters for YopE and SycE from purified pYV40 intoKpnI/HindIII site of pBad-MycHisA (Invitrogen). Additional modificationsinclude removal of the NcoI/BglII fragment of pBad-MycHisA by digest,Klenow fragment treatment and religation. Further at the 3′ end ofYopE₁₋₁₃₈ the following cleavage sites were added:XbaI-XhoI-BstBI-(HindIII). pBad_Si1 is equal to pBad_Si2 but encodesEGFP amplified from pEGFP-C1 (Clontech) in the NcoI/BglII site under theArabinose inducible promoter. Equally preferred is the use of modifiedversions of the endogenous Yersinia virulence plasmid pYV encodingheterologous proteins as fusions to a T3SS signal sequence.

A preferred vector e.g. a preferred expression vector for Salmonella isselected from the group consisting of pSi_266, pSi_267, pSi_268 andpSi_269. Plasmids pSi_266, pSi_267, pSi_268 and pSi_269 containing thecorresponding endogenous promoter and the SteA₁₋₂₀ fragment (pSi_266),the full length SteA sequence (pSi_267), the SopE₁₋₈₁ fragment (pSi_268)or the SopE₁₋₁₀₅ fragment (pSi_269) were amplified from S. entericaSL1344 genomic DNA and cloned into NcoI/KpnI site of pBad-MycHisA(Invitrogen).

The vectors of the instant invention may include other sequence elementssuch as a 3′ termination sequence (including a stop codon and a poly Asequence), or a gene conferring a drug resistance which allows theselection of transformants having received the instant vector.

The vectors of the present invention may be transformed by a number ofknown methods into the recombinant Gram-negative bacterial strains. Forthe purpose of the present invention, the methods of transformation forintroducing a vector include, but are not limited to, electroporation,calcium phosphate mediated transformation, conjugation, or combinationsthereof. For example, a vector can be transformed into a first bacteriastrain by a standard electroporation procedure. Subsequently, such avector can be transferred from the first bacteria strain into thedesired strain by conjugation, a process also called “mobilization”.Transformant (i.e., Gram-negative bacterial strains having taken up thevector) may be selected, e.g., with antibiotics. These techniques arewell known in the art. See, for example, [21].

In accordance with the present invention, the promoter of the expressionvector of the recombinant Gram-negative bacterial strain of theinvention can be a native promoter of a T3SS effector protein of therespective strain or a compatible bacterial strain or a promoter used inexpression vectors which are useful in e.g. Yersinia, Escherichia,Salmonella or Pseudomonas strain e.g pUC and pBad. Such promoters arethe T7 promoter, Plac promoter or Ara-bad promoter.

If the recombinant Gram-negative bacterial strain is a Yersinia strainthe promoter can be from a Yersinia virulon gene. A “Yersinia virulongene” refers to genes on the Yersinia pYV plasmid, the expression ofwhich is controlled both by temperature and by contact with a targetcell. Such genes include genes coding for elements of the secretionmachinery (the Ysc genes), genes coding for translocators (YopB, YopD,and LcrV), genes coding for the control elements (YopN, TyeA and LcrG),genes coding for T3SS effector chaperones (SycD, SycE, SycH, SycN, SycOand SycT), and genes coding for effectors (YopE, YopH, YopO/YpkA, YopM,YopT and YopP/YopJ) as well as other pYV encoded proteins as VirF andYadA.

In a preferred embodiment of the present invention, the promoter is thenative promoter of a T3SS functional effector encoding gene. If therecombinant Gram-negative bacterial strain is a Yersinia strain thepromoter is selected from any one of YopE, YopH, YopO/YpkA, YopM andYopP/YopJ. More preferably, the promoter is from YopE or SycE.

If the recombinant Gram-negative bacterial strain is a Salmonella strainthe promoter can be from SpiI or SpiII pathogenicity island or from aneffector protein elsewhere encoded. Such genes include genes coding forelements of the secretion machinery, genes coding for translocators,genes coding for the control elements, genes coding for T3SS effectorchaperones, and genes coding for effectors as well as other proteinsencoded by SPI-1 or SPI-2. In a preferred embodiment of the presentinvention, the promoter is the native promoter of a T3SS functionaleffector encoding gene. If the recombinant Gram-negative bacterialstrain is a Salmonella strain the promoter is selected from any one ofthe effector proteins. More preferably, the promoter is from SopE, InvBor SteA.

In a preferred embodiment the vector e.g. the expression vectorcomprises a DNA sequence encoding a protease cleavage site. Generationof a functional and generally applicable cleavage site allows cleavingoff the delivery signal after translocation. As the delivery signal caninterfere with correct localization and/or function of the translocatedprotein within the target cells the introduction of a protease cleavagesite between the delivery signal and the protein of interest providesfor the first time delivery of almost native proteins into eukaryoticcells. Preferably the protease cleavage site is an amino acid motifwhich is cleaved by a protease or the catalytic domains thereof selectedfrom the group consisting of enterokinase (light chain),enteropeptidase, prescission protease, human rhinovirus protease 3C, TEVprotease, TVMV protease, FactorXa protease and thrombin, more preferablyan amino acid motif which is cleaved by TEV protease. Equally preferablethe protease cleavage site is an amino acid motif which is cleaved by aprotease or the catalytic domains thereof selected from the groupconsisting of enterokinase (light chain), enteropeptidase, prescissionprotease, human rhinovirus protease 3C, TEV protease, TVMV protease,FactorXa protease, ubiquitin processing protease, calledDeubiquitinating enzymes, and thrombin. Most preferred is an amino acidmotif which is cleaved by TEV protease or by an ubiquitin processingprotease.

Thus in a further embodiment of the present invention, the heterologousprotein is cleaved from the delivery signal from a bacterial T3SSeffector protein by a protease. Preferred methods of cleavage aremethods wherein:

a) the protease is translocated into the eukaryotic cell by arecombinant Gram-negative bacterial strain as described herein whichexpresses a fusion protein which comprises the delivery signal from thebacterial T3SS effector protein and the protease as heterologousprotein; orb) the protease is expressed constitutively or transiently in theeukaryotic cell. Usually the recombinant Gram-negative bacterial strainused to deliver a desired protein into a eukaryotic cell and therecombinant Gram-negative bacterial strain translocating the proteaseinto the eukaryotic cell are different.

In one embodiment of the present invention the vector comprises afurther DNA sequence encoding a labelling molecule or an acceptor sitefor a labelling molecule.

The further DNA sequence encoding a labelling molecule or an acceptorsite for a labelling molecule is usually fused to the 5′ end or to the3′ end of the second DNA sequence. A preferred labelling molecule or anacceptor site for a labelling molecule is selected from the groupconsisting of enhanced green fluourescent protein (EGFP), coumarin,coumarin ligase acceptor site, resorufin, resurofin ligase acceptorsite, the tetra-Cysteine motif in use with FlAsH/ReAsH dye (lifetechnologies). Most preferred is resorufin and a resurofin ligaseacceptor site or EGFP. The use of a labelling molecule or an acceptorsite for a labelling molecule will lead to the attachment of a labellingmolecule to the heterologous protein of interest, which will then bedelivered as such into the eukaryotic cell and enables tracking of theprotein by e.g. live cell microscopy.

In one embodiment of the present invention the vector comprises afurther DNA sequence encoding a peptide tag. The further DNA sequenceencoding a peptide tag is usually fused to the 5′ end or to the 3′ endof the second DNA sequence. A preferred peptide tag is selected from thegroup consisting of Myc-tag, His-tag, Flag-tag, HA tag, Strep tag or V5tag or a combination of two or more tags out of these groups. Mostpreferred is Myc-tag, Flag-tag, His-tag and combined Myc- and His-tags.The use of a peptide tag will lead to traceability of the tagged proteine.g by immunofluorescence or Western blotting using anti-tag antibodies.Further, the use of a peptide tag allows affinity purification of thedesired protein either after secretion into the culture supernatant orafter translocation into eukaryotic cells, in both cases using apurification method suiting the corresponding tag (e.g. metal-chelateaffinity purification in use with a His-tag or anti-Flag antibody basedpurification in use with the Flag-tag).

In one embodiment of the present invention the vector comprises afurther DNA sequence encoding a nuclear localization signal (NLS). Thefurther DNA sequence encoding a nuclear localization signal (NLS) isusually fused to the 5′ end or to the 3′ end of the second DNA sequencewherein said further DNA sequence encodes a nuclear localization signal(NLS). A preferred NLS is selected from the group consisting of SV40large T-antigen NLS and derivates thereof [34] as well as other viralNLS. Most preferred is SV40 large T-antigen NLS and derivates thereof.

In one embodiment of the present invention the vector comprises amultiple cloning site. The multiple cloning site is usually located atthe 3′ end of the first DNA sequence and/or at the 5′ end or 3′ end ofthe second DNA sequence. One or more than one multiple cloning sites canbe comprised by the vector. A preferred multiple cloning site isselected from the group of restriction enzymes consisting of XhoI, XbaI,HindIII, NcoI, NotI, EcoRI, EcoRV, BamHI, NheI, SacI, SalI, BstBI. Mostpreferred is XbaI, XhoI, BstBI and HindIII.

The fused protein expressed from the first and second and optional thirdDNA sequences of the vector is also termed as a “fusion protein” or a“hybrid protein”, i.e., a fused protein or hybrid of delivery signal andrepeated domains of a heterologous protein or two or more domains ofdifferent heterologous proteins.

The present invention contemplates a method for delivering repeateddomains of a heterologous protein or two or more domains of differentheterologous proteins as hereinabove described into eukaryotic cells incell culture as well as in-vivo.

Thus in one embodiment the method for delivering repeated domains of aheterologous protein or two or more domains of different heterologousproteins comprises

i) culturing the Gram-negative bacterial strain as described herein;

ii) contacting a eukaryotic cell with the Gram-negative bacterial strainof i) wherein a fusion protein which comprises a delivery signal from abacterial T3SS effector protein and the repeated domains of aheterologous protein or the two or more domains of differentheterologous proteins is expressed by the Gram-negative bacterial strainand is translocated into the eukaryotic cell; and optionallyiii) cleaving the fusion protein so that the repeated domains of aheterologous protein or the two or more domains of differentheterologous proteins are cleaved from the delivery signal from thebacterial T3SS effector protein.

In some embodiments at least two fusion proteins which comprises each adelivery signal from a bacterial effector protein and repeated domainsof a heterologous protein or the two or more domains of differentheterologous proteins are expressed by the recombinant virulenceattenuated Gram-negative bacterial strain and are translocated into theeukaryotic cell by the methods of the present inventions.

The recombinant Gram-negative bacterial strain can be cultured so that afusion protein is expressed which comprises the delivery signal from thebacterial T3SS effector protein and the repeated domains of aheterologous protein or the two or more domains of differentheterologous proteins according to methods known in the art (e.g. FDA,Bacteriological Analytical Manual (BAM), chapter 8: Yersiniaenterocolitica). Preferably the recombinant Gram-negative bacterialstrain can be cultured in Brain Heart infusion broth e.g. at 28° C. Forinduction of expression of T3SS and e.g. YopE/SycE promoter dependentgenes, bacteria can be grown at 37° C.

In a preferred embodiment, the eukaryotic cell is contacted with twoGram-negative bacterial strains of i), wherein the first Gram-negativebacterial strain expresses a first fusion protein which comprises thedelivery signal from the bacterial T3SS effector protein and repeateddomains of a heterologous protein or two or more domains of differentheterologous proteins and the second Gram-negative bacterial strainexpresses a second fusion protein which comprises the delivery signalfrom the bacterial T3SS effector protein and repeated domains of asecond heterologous protein or two or more domains of a different secondheterologous proteins of a second heterologous protein, so that thefirst and the second fusion protein are translocated into the eukaryoticcell. This embodiment provided for co-infection of e.g eukaryotic cellswith two bacterial strains as a valid method to deliver e.g. twodifferent hybrid proteins into single cells to address their functionalinteraction.

Those skilled in the art can also use a number of assays to determinewhether the delivery of a fusion protein is successful. For example, thefusion protein may be detected via immunofluorescence using antibodiesrecognizing a fused tag (like Myc-tag). The determination can also bebased on the enzymatic activity of the protein being delivered.

The present invention contemplates a wide range of eukaryotic cells thatmay be targeted by the instant recombinant Gram-negative bacterialstrain e.g. Hi-5 (BTI-TN-5B1-4; life technologies B855-02), HeLa cells,e.g. HeLa Ccl2 (as ATCC No. CCL-2), fibroblast cells, e.g. 3T3fibroblast cells (as ATCC No. CCL-92) or Mef (as ATCC No. SCRC-1040),Hek (as ATCC No. CRL-1573), HUVECs (as ATCC No. PCS-100-013), CHO (asATCC No. CCL-61), Jurkat (as ATCC No. TIB-152), Sf-9 (as ATCC No.CRL-1711), HepG2 (as ATCC No. HB-8065), Vero (as ATCC No. CCL-81), MDCK(as ATCC No. CCL-34), THP-1 (as ATCC No. TIB-202), J774 (as ATCC No.TIB-67), RAW (as ATCC No. TIB-71), Caco2 (as ATCC No. HTB-37), NCI celllines (as ATCC No. HTB-182), DU145 (as ATCC No. HTB-81), Lncap (as ATCCNo. CRL-1740), MCF-7 (as ATCC No. HTB-22), MDA-MB cell lines (as ATCCNo. HTB-128), PC3 (as ATCC No. CRL-1435), T47D (as ATCC No. CRL-2865),A549 (as ATCC No. CCL-185), U87 (as ATCC No. HTB-14), SHSY5Y (as ATCCNo. CRL-2266s), Ea.Hy926 (as ATCC No. CRL-2922), Saos-2 (as ATCC No.HTBH-85), 4T1 (as ATCC No. CRL-2539), B16F10 (as ATCC No. CRL-6475), orprimary human hepatocytes (as life technologies HMCPIS), preferablyHeLa, Hek, HUVECs, 3T3, CHO, Jurkat, Sf-9, HepG2 Vero, THP-1, Caco2,Mef, A549, 4T1, B16F10 and primary human hepatocytes and most preferablyHeLa, Hek, HUVECs, 3T3, CHO, Jurkat, THP-1, A549 and Mef. By “target”,is meant the extracellular adhesion of the recombinant Gram-negativebacterial strain to a eukaryotic cell.

In accordance with the present invention, the delivery of a protein canbe achieved by contacting a eukaryotic cell with a recombinantGram-negative bacterial strain under appropriate conditions. Variousreferences and techniques are conventionally available for those skilledin the art regarding the conditions for inducing the expression andtranslocation of virulon genes, including the desired temperature, Ca⁺⁺concentration, addition of inducers as Congo Red, manners in which therecombinant Gram-negative bacterial strain and target cells are mixed,and the like. See, for example, [35]. The conditions may vary dependingon the type of eukaryotic cells to be targeted and the recombinantbacterial strain to be used. Such variations can be addressed by thoseskilled in the art using conventional techniques.

Those skilled in the art can also use a number of assays to determinewhether the delivery of a fusion protein is successful. For example, thefusion protein may be detected via immunofluorescence using antibodiesrecognizing a fused tag (like Myc-tag). The determination can also bebased on the enzymatic activity of the protein being delivered, e.g.,the assay described by [21].

In one embodiment the present invention provides the recombinantGram-negative bacterial strain as described herein for use in medicine.

In one embodiment the present invention provides the recombinantGram-negative bacterial strain as described herein for use in thedelivery of repeated domains of a heterologous protein or two or moredomains of different heterologous proteins as a medicament or as avaccine to a subject. The repeated domains of a heterologous protein orthe two or more domains of different heterologous proteins can bedelivered to a subject as a vaccine by contacting the Gram-negativebacterial strain with eukaryotic cells, e.g. with a living animal invivo so that the repeated domains of a heterologous protein or the twoor more domains of different heterologous proteins are translocated intothe living animal which then produces antibodies against the repeateddomains of a heterologous protein or the two or more domains ofdifferent heterologous proteins. The antibodies produced can be directlyused or be isolated and purified and used in diagnosis, in research useas well as in therapy. The B-cells producing the antibodies or thetherein contained DNA sequence can be used for further production ofspecific antibodies for use in diagnosis, in research use as well as intherapy.

In one embodiment the present invention provides a method for deliveringrepeated domains of a heterologous protein or the two or more domains ofdifferent heterologous proteins, wherein the repeated domains of aheterologous protein or two or more domains of different heterologousproteins are delivered in vitro into a eukaryotic cell.

In a further embodiment the present invention provides a method fordelivering repeated domains of a heterologous protein or two or moredomains of different heterologous proteins, wherein the eukaryotic cellis a living animal wherein the living animal is contacted with theGram-negative bacterial strain in vivo so that a fusion protein istranslocated into the living animal. The preferred animal is a mammal,more preferably a human being.

In a further embodiment the present invention provides the use of therecombinant Gram-negative bacterial strain as described supre for HighThroughput Screenings of inhibitors for a cellular pathway or eventtriggered by the translocated heterologous protein(s).

In a further aspect the present invention provides a library ofGram-negative bacterial strains, wherein the the repeated domains of aheterologous protein or the two or more domains of differentheterologous proteins encoded by the second DNA sequence of theexpression vector of the Gram-negative bacterial strains are domains ofa human or murine protein, preferably a domain of a human protein and,wherein each domain of a human or murine protein expressed by aGram-negative bacterial strain is different in amino acid sequence. Ascloning vector for expression the above described expression vectors canbe used.

In a further aspect the present invention provides a kit comprising avector as described herein and a bacterial strain expressing andsecreting a protease capable of cleaving the protease cleavage sitecomprised by the vector. A particular useful vector is a vector for usein combination with the bacterial strain to deliver a desired proteininto eukaryotic cells as described above, wherein the vector comprisesin the 5′ to 3′ direction:

a promoter;

a first DNA sequence encoding a delivery signal from a bacterial T3SSeffector protein, operably linked to said promoter;

a second DNA sequence encoding repeated domains of a heterologousprotein or two or more domains of different heterologous proteins fusedin frame to the 3′ end of said first DNA sequence,

wherein the heterologous proteins are selected from the group consistingof proteins involved in apoptosis or apoptosis regulation, cell cycleregulators, ankyrin repeat proteins, cell signaling proteins, reporterproteins, transcription factors, proteases, small GTPases, GPCR relatedproteins, nanobody fusion constructs and nanobodies, bacterial T3SSeffectors, bacterial T4SS effectors and viral proteins.

EXAMPLES Example 1

A) Materials and Methods

Bacterial strains and growth conditions. The strains used in this studyare listed in FIGS. 15A to N. E. coli Top10, used for plasmidpurification and cloning, and E. coli Sm10λ pir, used for conjugation,as well as E. coli BW19610 [36], used to propagate pKNG101, wereroutinely grown on LB agar plates and in LB broth at 37° C. Ampicillinwas used at a concentration of 200 μg/ml (Yersinia) or 100 μg/ml (E.coli) to select for expression vectors. Streptomycin was used at aconcentration of 100 μg/ml to select for suicide vectors. Y.enterocolitica MRS40 [22] a non Ampicillin resistant E40-derivate [21]and strains derived thereof were routinely grown on Brain Heart Infusion(BHI; Difco) at RT. To all Y. enterocolitica strains Nalidixic acid wasadded (35 μg/ml) and all Y. enterocolitica asd strains were additionallysupplemented with 100 μg/ml meso-2,6-Diaminopimelic acid (mDAP, SigmaAldrich). S. enterica SL1344 were routinely grown on LB agar plates andin LB broth at 37° C. Ampicillin was used at a concentration of 100μg/ml to select for expression vectors in S. enterica.

Genetic manipulations of Y. enterocolitica. Genetic manipulations of Y.enterocolitica has been described [37,38]. Briefly, mutators formodification or deletion of genes in the pYV plasmids or on thechromosome were constructed by 2-fragment overlapping PCR using purifiedpYV40 plasmid or genomic DNA as template, leading to 200-250 bp offlanking sequences on both sides of the deleted or modified part of therespective gene. Resulting fragments were cloned in pKNG101 [33] in E.coli BW19610 [36]. Sequence verified plasmids were transformed into E.coli Sm10λ pir, from where plasmids were mobilized into thecorresponding Y. enterocolitica strain. Mutants carrying the integratedvector were propagated for several generations without selectionpressure. Then sucrose was used to select for clones that have lost thevector. Finally mutants were identified by colony PCR. Specific mutators(pSi_408, pSi_419) are listed in Table III.

Construction of plasmids. Plasmid pBad_Si2 or pBad_Si1 (FIG. 10 ) wereused for cloning of fusion proteins with the N-terminal 138 amino acidsof YopE (SEQ ID No. 2). pBad_Si2 was constructed by cloning of theSycE-YopE₁₋₁₃₈ fragment containing endogenous promoters for YopE andSycE from purified pYV40 into KpnI/HindIII site of pBad-MycHisA(Invitrogen). Additional modifications include removal of the NcoI/BglIIfragment of pBad-MycHisA by digestion, Klenow fragment treatment andreligation. A bidirectional transcriptional terminator (BBa_B1006; iGEMfoundation) was cloned into KpnI cut and Klenow treated (pBad_Si2) orBglII cut site (pBad_Si1). Further at the 3′ end of YopE₁₋₁₃₈ thefollowing cleavage sites were added: XbaI-XhoI-BstBI-(HindIII) (FIG. 10B). pBad_Si1 is equal to pBad_Si2 but encodes EGFP amplified frompEGFP-C1 (Clontech) in the NcoI/BglII site under the Arabinose induciblepromoter. Plasmids pSi 266, pSi_267, pSi_268 and pSi_269 containing thecorresponding endogenous promoter and the SteA₁₋₂₀ fragment (pSi_266),the full length SteA sequence (pSi_267), the SopE₁₋₈₁ fragment (pSi_268)or the SopE₁₋₁₀₅ fragment (pSi_269) were amplified from S. entericaSL1344 genomic DNA and cloned into NcoI/KpnI site of pBad-MycHisA(Invitrogen).

Full length genes or fragments thereof were amplified with the specificprimers listed in Table I below and cloned as fusions to YopE₁₋₁₃₈ intoplasmid pBad_Si2 or in case of z-BIM (SEQ ID No. 21) into pBad_Si1 (seeTable II below). For fusion to SteA or SopE, synthetic DNA constructswere cleaved by KpnI/HindII and cloned into pSi_266, pSi_267, pSi_268 orpSi_269 respectively. In case of genes of bacterial species, purifiedgenomic DNA was used as template (S. flexneri M90T, Salmonella entericasubsp. enterica serovar Typhimurium SL1344, Bartonella henselae ATCC49882). For human genes a universal cDNA library (Clontech) was used ifnot otherwise stated (FIGS. 15A to N), zebrafish genes were amplifiedfrom a cDNA library (a kind gift of M. Affolter). Ligated plasmids werecloned in E. coli Top10. Sequenced plasmids were electroporated into thedesired Y. enterocolitica or S. enterica strain using settings as forstandard E. coli electroporation.

TABLE I (Primer Nr. Si_: Sequence)285: CATACCATGGGAGTGAGCAAGGGCGAG (SEQ ID NO: 44)286: GGAAGATCTttACTTGTACAGCTCGTCCAT (SEQ ID NO:  45)287: CGGGGTACCTCAACTAAATGACCGTGGTG (SEQ ID NO: 46)288: GTTAAAGCTTttcgaatctagactcgagCGTGGCGAACTGGTC (SEQ ID NO: 47)292: CAGTctcgagCAAATTCTAAACAAAATACTTCCAC (SEQ ID NO: 48)293: cagtTTCGAATTAATTTGTATTGCTTTGACGG (SEQ ID NO: 49)296: CAGTctcgagACTAACATAACACTATCCACCCAG (SEQ ID NO: 50)297: GTTAAAGCTTTCAGGAGGCATTCTGAAG (SEQ ID NO: 51)299: CAGTctcgagCAGGCCATCAAGTGTGTG (SEQ ID NO: 52)300: cagtTTCGAATCATTTTCTCTTCCTCTTCTTCA (SEQ ID  NO: 53)301: CAGTctcgagGCTGCCATCCGGAA (SEQ ID NO: 54)302: cagtTTCGAATCACAAGACAAGGCACCC (SEQ ID NO: 55)306: GTTAAAGCTTGGAGGCATTCTGAAGatacttatt (SEQ ID  NO: 56)307: CAGTctcgagCAAATACAGAGCTTCTATCACTCAG (SEQ ID NO: 57)308: GTTAAAGCTTTCAAGATGTGATTAATGAAGAAATG (SEQ ID NO: 58)317: cagtTTCGAACCCATAAAAAAGCCCTGTC (SEQ ID NO: 59)318: GTTAAAGCTTCTACTCTATCATCAAACGATAAAATGg (SEQ ID NO: 60)324: CAGTctcgagTTCACTCAAGAAACGCAAA (SEQ ID NO: 61)339: cagtTTCGAATTTTCTCTTCCTCTTCTTCAcg (SEQ ID NO: 62)341: cgtaTCTAGAAAAATGATGAAAATGGAGACTG (SEQ ID NO: 63)342: GTTAAAGCTTttaGCTGGAGACGGTGAC (SEQ ID NO: 64)346: CAGTctcgagTTCCAGATCCCAGAGTTTG (SEQ ID NO: 65)347: GTTAAAGCTTTCACTGGGAGGGGG (SEQ ID NO: 66)351: CAGTctcgagctcgagTTATCTACTCATAGAAACTACTTTTGCAG (SEQ ID NO: 67)352: cgcGGATCCtcagtgtctctgcggcatta (SEQ ID NO: 68)353: CATTTATTCCTCCTAGTTAGTCAcagcaactgctgctcctttc (SEQ ID NO: 69)354: gaaaggagcagcagttgctgTGACTAACTAGGAGGAATAAATG (SEQ ID NO: 70)355: cgattcacggattgctttctCATTATTCCCTCCAGGTACTA (SEQ ID NO: 71)356: TAGTACCTGGAGGGAATAATGagaaagcaatccgtgaatcg (SEQ ID NO: 72)357: cgtaTCTAGAcggetttaagtgcgacattc (SEQ ID NO: 73)364: cgtaTCTAGACTAAAGTATGAGGAGAGAAAATTGAA (SEQ ID NO: 74)365: GTTAAAGCTTTCAGCTTGCCGTCGT (SEQ ID NO: 75)367: CGTAtctagaGACCCGTTCCTGGTGC (SEQ ID NO: 76)369: cgtaTCTAGAccccccaagaagaagc (SEQ ID NO: 77)373: GTTAAAGCTTGCTGGAGACGGTGACC (SEQ ID NO: 78)386: CGTAtctagaTCAGGACGCTTCGGAGGTAG (SEQ ID NO: 79)387: CGTAtctagaATGGACTGTGAGGTCAACAA (SEQ ID NO: 80)389: CGTAtctagaGGCAACCGCAGCA (SEQ ID NO: 81)391: GTTAAAGCTTTCAGTCCATCCCATTTCTg (SEQ ID NO: 82)403: CGTAtctagatctggaatatccctggaca (SEQ ID NO: 83)406: GTTAAAGCTTgtctgtctcaatgccacagt (SEQ ID NO: 84)410: CAGTctcgagATGTCCGGGGTGGTg (SEQ ID NO: 85)413: cagtTTCGAATCACTGCAGCATGATGTC (SEQ ID NO: 86)417: CAGTctcgagAGTGGTGTTGATGATGACATG (SEQ ID NO:  87)420: cagtTTCGAATTAGTGATAAAAATAGAGTTCTTTTGTGAG  (SEQ ID NO: 88)423: CAGTctcgagATGCACATAACTAATTTGGGATT (SEQ ID  NO: 89)424: cagtTTCGAATTATACAAATGACGAATACCCTTT (SEQ ID NO: 90)425: GTTAAAGCTTttacaccttgcgcttcttcttgggcggGCTGGAGACGGTGAC (SEQ ID NO: 91)428: CGTAtctagaATGGACTTCAACAGGAACTTT (SEQ ID NO: 92)429: CGTAtctagaGGACATAGTCCACCAGCG (SEQ ID NO: 93)430: GTTAAAGCTTTCAGTTGGATCCGAAAAAC (SEQ ID NO: 94)433: CGTAtctagaGAATTAAAAAAAACACTCATCCCA (SEQ ID NO: 95)434: CGTAtctagaCCAAAGGCAAAAGCAAAAA (SEQ ID NO: 96)435: GTTAAAGCTTTTAGCTAGCCATGGCAAGC (SEQ ID NO: 97)436: CGTAtctagaATGCCCCGCCCC (SEQ ID NO: 98)437: GTTAAAGCTTCTACCCACCGTACTCGTCAAT (SEQ ID NO: 99)438: CGTAtctagaATGTCTGACACGTCCAGAGAG (SEQ ID NO: 100)439: GTTAAAGCTTTCATCTTCTTCGCAGGAAAAAG (SEQ ID NO: 101)445: cgcGGATCCttatgggttctcacagcaaaa (SEQ ID NO:  102)446: CATTTATTCCTCCTAGTTAGTCAaggcaacagccaatcaagag (SEQ ID NO: 103)447: ctettgattggctgttgcctTGACTAACTAGGAGGAATAAATG (SEQ ID NO: 104)448: ttgattgcagtgacatggtgCATTATTCCCTCCAGGTACTA (SEQ ID NO: 105)449: TAGTACCTGGAGGGAATAATGcaccatgtcactgcaatcaa (SEQ ID NO: 106)450: cgtaTCTAGAtagccgcagatgttggtatg (SEQ ID NO: 107)451: CGTAtctagaGATCAAGTCCAACTGGTGG (SEQ ID NO: 108)463: CAGTctcgaggaaagettgtttaaggggc (SEQ ID NO:  109)464: cagtTTCGAAttagcgacggcgacg (SEQ ID NO: 110)476: GTTAAAGCTTttACTTGTACAGCTCGTCCAT (SEQ ID NO: 111)477: CGTAtctagaGTGAGCAAGGGCGAG (SEQ ID NO: 112)478: CAGTctcgagATGGAAGATTATACCAAAATAGAGAAA (SEQ ID NO: 113)479: GTTAAAGCTTCTACATCTTCTTAATCTGATTGTCCa (SEQ ID NO: 114)482: CGTAtctagaATGGCGCTGCAGCt (SEQ ID NO: 115)483: GTTAAAGCTTTCAGTCATTGACAGGAATTTTg (SEQ ID NO: 116)486: CGTAtctagaATGGAGCCGGCGGCG (SEQ ID NO: 117)487: GTTAAAGCTTTCAATCGGGGATGTCTg (SEQ ID NO: 118)492: CGTAtctagaATGCGCGAGGAGAACAAGGG (SEQ ID NO: 119)493: GTTAAAGCTTTCAGTCCCCTGTGGCTGTGc (SEQ ID NO: 120)494: CGTAtctagaATGGCCGAGCCTTG (SEQ ID NO: 121)495: GTTAAAGCTTttaTTGAAGATTTGTGGCTCC (SEQ ID NO: 122)504: CGTAtctagaGAAAATCTGTATTTTCAAAGTGAAAATCTGTATTTTCAAAGTATGCCCCGCCCC (SEQ ID NO: 123)505: GTTAAAGCTTCCCACCGTACTCGTCAATtc (SEQ ID NO: 124)508: CGTAtctagaGAAAATCTGTATTTTCAAAGTGAAAATCTGTATTTTCAAAGTATGGCCGAGCCTTG (SEQ ID NO: 125)509: GTTAAAGCTTTTGAAGATTTGTGGCTCCc (SEQ ID NO:  126)511: CGTAtctagaGAAAATCTGTATTTTCAAAGTGAAAATCTGTATTTTCAAAGTGTGAGCAAGGGCGAG (SEQ ID NO: 127)512: CGTAtctagaGAAAATCTGTATTTTCAAAGTGAAAATCTGTATTTTCAAAGTCCGCCGAAAAAAAAACGTAAAGTTGTGAGCAAGGGCGAG (SEQ ID NO: 128)513: GTTAAAGCTTttAAACTTTACGTTTTTTTTTCGGCGGCTTGTACAGCTCGTCCAT (SEQ ID NO: 129)515: CGTAtctagaGAAAATCTGTATTTTCAAAGTGAAAATCTGTATTTTCAAAGTGATTATAAAGATGATGATGATAAAATGGCCGAGCCTTG (SEQ ID NO: 130)558: CGTATCTAGAATGACCAGTTTTGAAGATGC (SEQ ID NO: 131)559: GTTAAAGCTTTCATGACTCATTTTCATCCAT (SEQ ID NO: 132)561: CGTATCTAGAATGAGTCTCTTAAACTGTGAGAACAG (SEQ ID NO: 133)562: GTTAAAGCTTCTACACCCCCGCATCA (SEQ ID NO: 134)580: catgccatggATTTATGGTCATAGATATGACCTC (SEQ ID NO: 152)585: CAGTctcgagATGCAGATCTTCGTCAAGAC (SEQ ID NO: 197)586: GTTAAAGCTTgctagcttcgaaACCACCACGTAGACGTAAGAC (SEQ ID NO: 198)588: cagtTTCGAAGATTATAAAGATGATGATGATAAAATGGCCGAGCC TTG (SEQ ID NO: 199)612: CGGGGTACCatgaggtagcttatttcctgataaag (SEQ ID NO: 153)613: CGGGGTACCataattgtccaaatagttatggtagc (SEQ ID  NO: 154)614: catgccatggCGGCAAGGCTCCTC (SEQ ID NO: 155)615: cggggtaccTTTATTTGTCAACACTGCCC (SEQ ID NO:  156)616: cggggtaccTGCGGGGTCTTTACTCG (SEQ ID NO: 157)677: TTACTATTCGAAGAAATTATTCATAATATTGCCCGCCATCTGGCCCAAATTGGTGATGAAATGGATCATTAAGCTTGGAGTA (SEQ ID NO: 148)678: TACTCCAAGCTTAATGATCCATTTCATCACCAATTTGGGCCAGATGGCGGGCAATATTATGAATAATTTCTTCGAATAGTAA (SEQ ID NO: 149)682: TTACTACTCGAGAAAAAACTGAGCGAATGTCTGCGCCGCATTGGTGATGAACTGGATAGCTAAGCTTGGAGTA (SEQ ID NO: 150)683: TACTCCAAGCTTAGCTATCCAGTTCATCACCAATGCGGCGCAGACATTCGCTCAGTTTTTTCTCGAGTAGTAA (SEQ ID NO: 151)725: TTACTATTCGAAGAAATTATTCATAATATTGCC (SEQ ID  NO: 220)726: TACTCCAAGCTTACGGTTGAATATTATGATCCATTTCATCACCAATTTGG (SEQ ID NO: 221)727: TTACTATTCGAAGCCGGTGGTGCCGAAGAAATTATTCATAATATT GCCC (SEQ ID NO: 222)728: TACTCCAAGCTTAATGATCCATTTCATCA (SEQ ID NO:  223)733: TTACTACTCGAGGGTGCCATCGATGCCGAAGAAATTATTCATAATATTGCCCG (SEQ ID NO: 204)734: TACTCCTTCGAAGGCACCATGATCCATTTCATCACCAATTTGG (SEQ ID NO: 208)735: TACTCCTTCGAATTAATGATCCATTTCATCACCAATTTG (SEQ ID NO: 205)736: TTACTACTCGAGGGTGCCATCGATGCCAAAAAACTGAGCGAATGTCTGCG (SEQ ID NO: 206) 737: TACTCCTTCGAAGGCACCGCTATCCAGTTCATCACCAATG(SEQ ID NO: 224) 738: TACTCCTTCGAATTAGCTATCCAGTTCATCACCAATG (SEQ ID NO: 207)

TABLE II Cloned fusion proteins Protein to be Protein Resulting Primerdelivred by Seq. ID. Backbone plasmid Primers. Seq. ID T3SS No. plasmidname Si_Nr.: No. YopE1-138- 3 pBad- pBad_Si_1 285/286 44/45 and MycHisMycHisA (EGFP), 46/47 (Invitrogen) 287/288 (sycE- YopE1-138) YopE1-138-3 pBad- pBad_Si_2 287/288 46/47 MycHis MycHisA (sycE- (Invitrogen)YopE1-138) YopE1-138- 4 pBad_Si_2 pSi_16 292/293 48/49 IpgB1 YopE1-138-5 pBad_Si_2 pSi_20 296/297 50/51 SopE YopE1-138- 26 pBad_Si_2 pSi_22299/300 52/53 Rac1 Q61L YopE1-138- 27 pBad_Si_2 pSi_24 301/302 54/55RhoA Q61E YopE1-138- 135 pBad_Si_2 pSi_28 296/306 50/56 SopE-MycHisYopE1-138- 6 pBad_Si_2 pSi_30 307/308 57/58 SopB YopE1-138- 28 pBad_Si_2pSi_37 367/386 76/79 FADD YopE1-138- 7 pBad_Si_2 pSi_38 317/318 59/60OspF YopE1-138- 136 pBad_Si_2 pSi_43 324/351 61/67 BepG 715-endYopE1-138- 137 pBad_Si_2 pSi_51 299/339 52/62 Rac1 Q61L- MycHisYopE1-138- 32 pBad_Si_2 pSi_53 341/342 63/64 Slmb1-VhH4 YopE1-138-Bad 29pBad_Si_2 pSi_57 346/347 65/66 YopE1-138- 8 pBad_Si_2 pSi_64 364/36574/75 SptP YopE1-138- 33 pBad_Si_2 pSi_70 369/342 77/64 NLS-Slmb1- VhH4YopE1-138-Bid 24 pBad_Si_2 pSi_85 387/391 80/82 YopE1-138-t- 25pBad_Si_2 pSi_87 389/391 81/82 Bid YopE1-138- 22 pBad_Si_2 pSi_97403/406 83/84 Caspase3 p17 YopE1-138- 30 pBad_Si_2 pSi_103 410/413 85/86GPCR GNA12 YopE1-138- 23 pBad_Si_2 pSi_106 417/420 87/88 Caspase3 p10/12YopE1-138- 9 pBad_Si_2 pSi_111 423/424 89/90 IpgD YopE1-138- 34pBad_Si_2 pSi_112 341/425 63/91 Slmb1-VhH4- NLS YopE1-138-z-Bid 19pBad_Si_2 pSi_116 428/430 92/94 YopE1-138-z-t-Bid 20 pBad_Si_2 pSi_117429/430 93/94 YopE1-138- 11 pBad_Si_2 pSi_118 433/435 95/97 BepAE305-end YopE1-138- 10 pBad_Si_2 pSi_119 434/435 96/97 BepAYopE1-138-ET1 36 pBad_Si_2 pSi_120 436/437 98/99 YopE1-138-z- 21pbad_Si_1 pSi_121 438/439 100/101 BIM YopE1-138- 31 pBad_Si_2 pSi_124451/373 108/78 VhH4 nanobody recognizing EGFP YopE1-138- 42 pBad_Si_2pSi_132 463/464 109/110 TEV protease S219V YopE1-138- 37 pBad_Si_2pSi_140 477/476 112/111 EGFP YopE1-138- 14 pBad_Si_2 pSi_143 478/479113/114 Cdkl YopE1-138- 15 pBad_Si_2 pSi_145 482/483 115/116 Mad2YopE1-138- 16 pBad_Si_2 pSi_147 486/487 117/118 Ink4A YopE1-138- 17pBad_Si_2 pSi_150 492/493 119/120 Ink4B YopE1-138- 18 pBad_Si_2 pSi_151494/495 121/122 Ink4C YopE1-138- 13 pBad_Si_2 pSi_153 558/559 131/132TIFA YopE1-138-2x 41 pBad_Si_2 pSi_156 504/505 123/124 TEVsite - ET1YopE1-138- 39 pBad_Si_2 pSi_159 511/513 127/129 2xTEVsite EGFP-NLSYopE1-138- 38 pBad_Si_2 pSi_160 512/476 128/111 2xTEVsite NLS-EGFPYopE1-138-2x 40 pBad_Si_2 pSi_161 508/509 125/126 TEVsite INK4CYopE1-138-2x 43 pBad_Si_2 pSi_164 515/509 130/126 TEVsite - Flag - INK4CYopE1-138- 12 pBad_Si_2 pSi_166 561/562 133/134 murine Traf6YopE1-138-Y. 138 pBad_Si_2 pSi_318 677/678 148/149 enterocolitica codonoptimized murine tBid BH3 part YopE1-138-Y. 139 pBad_Si_2 pSi_322682/683 150/151 enterocolitica codon optimized murine Bax BH3 partpBad-MycHisA SteA1-20 140 pBad-MycHisA pSi_266 580/612 152/153(Invitrogen) SteA 141 pBad-MycHisA pSi_267 580/613 152/154 (Invitrogen)SopE1-81 142 pBad-MycHisA pSi_268 614/615 155/156 (Invitrogen) SopE1-105143 pBad-MycHisA pSi_269 614/616 155/157 (Invitrogen) SteA1-20-S. 144pSi_266 pSi_270 synthetic / enterica codon construct optimized murinetBid SteA-S. enterica 145 pSi_267 pSi_271 synthetic / codon optimizedconstruct murine tBid SopE1-81-S. 146 pSi_268 pSi_272 synthetic /enterica codon construct optimized murine tBid SopE1-105-S. 147 pSi_269pSi_273 synthetic / enterica codon construct optimized murine tBidYopE1-138-Y. 158 pBad_Si_2 pSi_362 745/746 172/173 enterocolitica codonoptimized Ink4A 84-103 YopE1-138-Y. 159 pBad_Si_2 pSi_363 747/748174/175 enterocolitica codon optimized p107/RBL1 657-662 (AAA02489.1)YopE1-138-Y. 160 pBad_Si_2 pSi_364 749/750 176/177 enterocolitica codonoptimized p21 141-160 (AAH13967.1) YopE1-138-Y. 161 pBad_Si_2 pSi_366753/754 178/179 enterocolitica codon optimized p21 145-160 (AAH13967.1)YopE1-138-Y. 162 pBad_Si_2 pSi_367 755/756 180/181 enterocolitica codonoptimized p21 17-33 (AAH13967.1) YopE1-138-Y. 163 pBad_Si_2 pSi_368757/758 182/183 enterocolitica codon optimized cyclin D2 139- 147(CAA48493.1) SteA-Ink4a- 164 pSi_267 pSi_333 703/704 184/185 MycHisSopE1-105- 165 pSi_269 pSi_334 703/704 184/185 Ink4a-MycHis SteA-Ink4c-166 pSi_267 pSi_335 PCR1: 186/187, MycHis pSi_267 188/189 705/706; PCR2:707/708; overlapping PCR: 705/708 SopE1-105- 167 pSi_269 pSi_336 PCR1:186/187, Ink4c-MycHis 705/706; 188/189 PCR2: 707/708; overlapping PCR:705/708 SteA-Mad2- 168 pSi_267 pSi_337 709/710 190/191 MycHis SopE1-105-169 pSi_269 pSi_338 709/710 190/191 Mad2-MycHis SteA-Cdk1- 170 pSi_267pSi_339 711/712 192/193 MycHis SopE1-105- 171 pSi_269 pSi_340 711/712192/193 Cdkl-MycHis YopE1-138-Y. 194 pBad_Si_2 pSi_315 synthetic /enterocolitica construct codon optimized murine tBid YopE1-138- 195pBad_Si_2 pSi_236 585/586 197/198 Ubiquitin YopE1-138- 196 pSi_236pSi_237_II 588/509 199/126 Ubiquitin-Flag- INK4C-MycHis 200 pBad_Si_2pSi_357 733/735 204/205 YopE1-138-(Y. enterocolitica codon optimizedmurine tBid BH3 part) ready for insertion of further domainsYopE1-138-(Y. 201 pBad_Si_2 pSi_358 736/738 206/207 enterocolitica codonoptimized murine BAX BH3 part) ready for insertion of further domainsYopE1-138-(Y. 202 pSi_357 pSi_371 733/734 204/208 enterocolitica codonoptimized murine tBid BH3 part)₂ YopE1-(138-Y. 203 pSi_358 pSi_373733/734 204/208 enterocolitica 209 pBad_Si_2 pSi_353 725/726 212/213codon optimized murine tBid BH3 part-Y. enterocolitica codon optimizedmurine BAX BH3 part YopE₁_138- codon optimized murine tBid BH3 extendedpart YopE₁₋₁₃₈-10 Aa 210 pBad_Si_2 pSi_354 727/728 214/215 linker - Y.enterocolitica codon optimized murine tBid BH3 part YopE1-(138-Y. 211pSI_357 pSi_374 736/737 206/216 enterocolitica codon optimized murineBax BH3 part- Y. enterocolitica codon optimized murine tBid BH3 part

TABLE III Mutators for genetic modification Mutator/ To be BackboneResulting Primers Primers used with Construct inserted onto: plasmidplasmid name Si_Nr.: Seq. Id No. parent strain YopE₁₋₁₃₈- pYV pKNG101pSi_408 Synthetic / / tBID BH3 gene YopE₁₋₁₃₈- pYV pKNG101 pSi_419Synthetic / Strain (tBID BH3)₂ gene mutated with pSi_408

Yop secretion. Induction of the yop regulon was performed by shiftingthe culture to 37° C. in BHI-Ox (secretion-permissive conditions) [39].As carbon source glucose was added (4 mg/ml).

Total cell and supernatant fractions were separated by centrifugation at20 800 g for 10 min at 4° C. The cell pellet was taken as total cellfraction. Proteins in the supernatant were precipitated withtrichloroacetic acid 10% (w/v) final for 1 h at 4° C. Aftercentrifugation (20 800 g for 15 min) and removal of the supernatant, theresulting pellet was washed in ice-cold Acetone over-night. The sampleswere centrifuged again, the supernatant was discarded and the pellet wasair-dried and resuspended in 1×SDS loading dye.

Secreted proteins were analysed by SDS-PAGE; in each case, proteinssecreted by 3×10⁸ bacteria were loaded per lane. Detection of specificsecreted proteins by immunoblotting was performed using 12.5% SDS-PAGEgels. For detection of proteins in total cells, 2×10⁸ bacteria wereloaded per lane, if not stated otherwise, and proteins were separated on12.5% SDS-PAGE gels before detection by immunoblotting.

Immunoblotting was carried out using rat monoclonal antibodies againstYopE (MIPA193-13A9; 1:1000, [40]). The antiserum was preabsorbed twiceovernight against Y. enterocolitica ΔHOPEMT asd to reduce backgroundstaining. Detection was performed with secondary antibodies directedagainst rat antibodies and conjugated to horseradish peroxidase (1:5000;Southern biotech), before development with ECL chemiluminescentsubstrate (LumiGlo, KPM).

Cell culture and infections. HeLa Cc12, swiss 3T3 fibroblast cells, 4T1,B16F10 and D2A1 were cultured in Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% FCS and 2 mM L-Glutamine (cDMEM). HUVECswere isolated and cultivated as described [41]. Jurkat and 4T1 cellswere cultured in RPMI 1640 supplemented with 10% FCS and 2 mML-Glutamine. Y. enterocolitica were grown in BHI with additivesovernight at RT, diluted in fresh BHI to an OD₆₀₀ of 0.2 and grown for 2h at RT before a temperature shift to a 37° C. waterbath shaker forfurther 30 min or for 1 h in case of delivery of EGFP. Finally, thebacteria were collected by centrifugation (6000 rcf, 30 sec) and washedonce with DMEM supplemented with 10 mM HEPES and 2 mM L-glutamine. S.enterica were grown in LB with additives overnight at 37° C. and eitherdiluted 1:40 in fresh LB and grown for 2.5 h at 37° C. (SpiI T3SSinducting conditions) or the overnight culture was further incubated at37° C. (SpiII T3SS inducing conditions). Finally, the bacteria werecollected by centrifugation (6000 rcf, 30 sec) and washed once with DMEMsupplemented with 10 mM HEPES and 2 mM L-glutamine. Cells seeded in96-well (for Immunofluorescence) or 6-well (for Western blotting) plateswere infected at indicated MOIs in DMEM supplemented with 10 mM HEPESand 2 mM L-glutamine. After adding bacteria, plates were centrifuged for1 min at 1750 rpm and placed at 37° C. for indicated time periods.Extracellular bacteria were killed by gentamicin (100 mg/ml) ifindicated. In case of immunofluorescence analysis, infection assays werestopped by 4% PFA fixation. For Western blot analysis cells were washedtwice with ice-cold PBS and Phospho-safe lysis buffer (Novagen) wasadded to lyse the cells. After incubation on ice, the cells werecentrifuged (16 000 rcf, 25 min, 4° C.). Supernatants were collected andanalyzed for total protein content by Bradford BCA assay (Pierce) beforeSDS PAGE and Western blotting using anti-Phospho-Akt (Ser473 and T308,both Cell Signaling), anti-Actin (Millipore), Anti-Bid (Cell Signaling),anti-Myc (Santa Cruz), anti-p38 (Cell Signaling), anti-phospho-p-38(Thr180/Tyr182; Cell Signaling), anti-Caspase-3 p17 (Cell Signaling) andanti-Ink4C (Cell Signaling) antibody.

Secretion analysis with S. enterica. For induction of protein secretionby S. enterica, S. enterica were cultivated overnight in LB containing0.3 M NaCl on an orbital shaker (set to 150 rpm). S. enterica were thendiluted 1:50 in fresh LB containing 0.3 M NaCl and grown for 4 h at 37°C. without shaking.

Total cell and supernatant fractions were separated by centrifugation at20 800 g for 20 min at 4° C. The cell pellet was taken as total cellfraction. Proteins in the supernatant were precipitated withtrichloroacetic acid 10% (w/v) final for 1 h at 4° C. Aftercentrifugation (20 800 g for 15 min) and removal of the supernatant, theresulting pellet was washed in ice-cold Acetone over-night. The sampleswere centrifuged again, the supernatant was discarded and the pellet wasair-dried and resuspended in 1×SDS loading dye.

Secreted proteins were analysed by SDS-PAGE; in each case, proteinssecreted by 3×108 bacteria were loaded per lane. Detection of specificsecreted proteins by immunoblotting was performed using 12.5% SDS-PAGEgels. For detection of proteins in total cells, 2×10⁸ bacteria wereloaded per lane, if not stated otherwise, and proteins were separated on12.5% SDS-PAGE gels before detection by immunoblotting. Immunoblottingwas carried out using anti-Myc (Santa Cruz) antibody.

Western blotting of T3SS translocated proteins from infected cells. HeLacells in 6-well plates were infected at an MOI of 100 as describedabove. In case of coinfection with the TEV protease translocating Y.enterocolitica strain, the OD₆₀₀ of the strains was set and the twobacterial suspensions were mixed in a tube at a ratio of 1:1 (if nototherwise indicated) before addition to the cells. At the end of theinfection, the cells were washed twice with ice-cold PBS and collectedby scraping in a small volume of ice-cold PBS. After centrifugation (16000 rcf, 5 min, 4° C.) the pellet was dissolved in 0.002% digitoninsupplemented with a protease inhibitor cocktail (Roche complete, Roche).The dissolved pellets were incubated for 5 minutes on ice and thencentrifuged (16 000 rcf, 25 min, 4° C.). Supernatants were collected andanalyzed for total protein content by Bradford BCA assay (Pierce) beforeSDS PAGE and Western blotting using an anti-Myc (Santa Cruz, 9E11) oranti-Ink4C (Cell Signaling) antibody.

Immunofluorescence. Cell seeded in 96-well plates (Corning) wereinfected as described above and after fixation with 4% PFA the cellswere washed three times with PBS. The wells were then blocked using 5%goat serum in PBS 0.3% Triton X-100 for 1 h at RT. The primary antibody(anti-Myc, Santa Cruz, 1:100) was diluted in PBS with 1% BSA and 0.3%Triton X-100 and cells were incubated overnight at 4° C. Cells werewashed 4 times with PBS before the secondary antibody (AF 488anti-mouse, life technologies, 1:250) diluted in PBS with 1% BSA and0.3% Triton X-100 was added. If needed Hoechst DNA staining (lifetechnologies, 1:2500) and/or actin staining (Dy647-Phalloidin, DyeOmics)were included. In some cases only the DNA and/or actin stain was applieddirectly after washing the PFA off. Cells were incubated for 1 h at RT,washed three times with PBS and analyzed by automated image analysis asdescribed below.

Automated Microscopy and Image Analysis. Images were automaticallyacquired with an ImageXpress Micro (Molecular devices, Sunnyvale, USA).Quantification of anti-Myc staining intensities was performed usingMetaXpress (Molecular devices, Sunnyvale, USA). Regions within cellsexcluding nuclear regions and regions containing bacteria were manuallychosen (circles with an area of 40 pixels) and average intensity wasrecorded.

TNFα stimulation and Western blotting of phospho-p38. HeLa cells seededin 6-well plates were infected with an MOI of 100 as described above. 30min p.i Gentamicin was added and 45 min p.i. TNFa was added (10 ng/ml).1 h 15 min p.i. cells were washed twice with ice-cold PBS andPhospho-safe lysis buffer (Novagen) was added to lyse the cells. Afterincubation on ice, the cells were centrifuged (16 000 rcf, 25 min, 4°C.). Supernatants were collected and analyzed for total protein contentby Bradford BCA assay (Pierce) before SDS PAGE and Western blottingusing an anti-Phospho-p38, total p38 antibodies (Cell Signaling) andanti-Actin antibody (Millipore).

cAMP level determination of infected HeLa cells. HeLa cells seeded in96-well plates were infected as described above. 30 min before theinfection cDMEM was changed to DMEM supplemented with 10 mM HEPES and 2mM L-glutamine and 100 uM 3-Isobutyl-1-methylxanthin (IBMX, SigmaAldrich). 60 min p.i. Gentamicin was added and cells were furtherincubated at 37° C. for another 90 min. Determination of cAMP wasperformed using a competitive ELISA according to the manufacturersinstructions (Amersham, cAMP Biotrak, RPN225). As a positive controlindicated amount of cholera toxin (C8052, Sigma Aldrich) was added for 1h to cells in DMEM supplemented with 10 mM HEPES and 2 mM L-glutamineand 100 uM IBMX.

Zebrafish Embryo Infections, Imaging and Automated Image Quantification.All animal experiments were performed according to approved guidelines.Zebrafish were maintained at standard conditions [42]. Embryos werestaged by hours postfertilization (hpf) at 28.5° C. [43]. The followingzebrafish lines were used in this study: wild type fish (AB/EK andEK/TL). Infection protocol followed guidelines given in [44]. 12 hpfembryos were maintained in E3 medium containing 0.2 mM N-phenylthiourea(PTU) to prevent pigment formation. 2 days postfertilization (dpf)embryos were anesthetized by 0.2 mg/ml Tricaine and aligned on 1% agarplates in E3 using a hair loop tool [44]. Y. enterocolitica were grownin BHI supplemented with 0.4% Arabinose and antibiotics and mDapovernight at RT, diluted in fresh BHI with 0.5% Arabinose and otheradditives to an OD₆₀₀ of 0.2 and grown for 2 h at RT before atemperature shift to a 37° C. waterbath shaker for further 45 min.Finally, the bacteria were collected by centrifugation (6000 rcf, 30sec) and washed once with PBS. The OD₆₀₀ was set to 2 in PBS containingmDAP. 1-2 nL of this suspension were injected into the hindbrain ofaligned zebrafish embryos using an Femtojet Microinjector (Eppendorf)using Femtotips II (Eppendorf), where the tip of the needle had beenbroken off with fine tweezers. The injection time was set to 0.2 s andthe compensation pressure to 15 hPa (Eppendorf, Femtojet) and theinjection pressure was adjusted between 600 and 800 hPa. Drop size andthus the inoculum was checked by microscopy and by control plating.Following microinjection the fish were collected in E3 containingTricaine and PTU and incubated for 30 min at 37° C. and incubated forfurther 5 h at 28° C. A fluorescence binocular (Leica) was used toobserve bacterial EGFP fluorescence 1 h post infection in zebrafishhindbrains, and embryos that are not properly injected were discarded.At the end of the infection, fish were fixed with 2% ice-cold PFA for 1h on ice and further with fresh ice-cold PFA overnight at 4° C. Antibodystaining was performed as described previously [45,46]. Briefly, embryoswere washed 4 times with PBS 0.1% Tween for 5 min each wash andpermeabilized with PBS-T+0.5% Triton X-100 for 30 min at RT. Embryoswere blocked in blocking solution (PBS 0.1% Tween 0.1% TritonX-100 5%goat serum and 1% BSA) at 4° C. overnight. Antibody (Cleaved Caspase-3(Asp175), Cell Signaling) was diluted 1:100 in blocking solution andincubated under shaking at 4° C. in the dark. Fish were washed 7 timeswith PBS 0.1% Tween for 30 min before the secondary antibody (goatanti-rabbit AF647, Invitrogen, 1:500) diluted in blocking solution wasadded and incubated at 4° C. overnight. Larvae were washed with PBS 0.1%Tween four times 30 min at 4° C. and once overnight and further washed3-4 times. Images were taken with Leica TCS SP5 confocal microscopeusing a 40× water immersion objective. Images were analyzed using Imaris(Bitplane) and Image J software (world wide web address:imagej.nih.gov/ij/).

Image analysis (on n=14 for pBad_Si2 or n=19 for z-BIM) was performedvia CellProfiler [47] on maximum intensity z projections of recordedz-stack images. Briefly, bacteria were detected via the GFP channel.Around each area of a bacterial spot a circle with a radius of 10 pixelswas created. Overlapping regions were separated equally among theconnecting members. In those areas closely surrounding bacteria, theCaspase 3 p17 staining intensity was measured.

Sample Preparation for Phosphoproteomics. For each condition, two 6-wellplates of HeLa CCL-2 cells were grown to confluency. Cells were infectedfor 30 min as described above. At the indicated time-points, the plateswere put on ice and washed twice with ice-cold PBS. Samples were thencollected in urea solution [8 M Urea (AppliChem), 0.1 MAmmoniumbicarbonate (Sigma), 0.1% RapiGest (Waters), 1× PhosSTOP(Roche)]. The samples were briefly vortexed, sonicated at 4° C.(Hielscher), shaked for 5 min on a thermomixer (Eppendorf) andcentrifuged for 20 min at 4° C. and 16′000 g. Supernatants werecollected and stored at −80° C. for further processing. BCA ProteinAssay (Pierce) was used to measure protein concentration.

Phosphopeptide Enrichment. Disulfide bonds were reduced withtris(2-carboxyethyl)phosphine at a final concentration of 10 mM at 37°C. for 1 h. Free thiols were alkylated with 20 mM iodoacetamide (Sigma)at room temperature for 30 min in the dark. The excess of iodoacetamidewas quenched with N-acetyl cysteine at a final concentration of 25 mMfor 10 min at room temperature. Lys-C endopeptidase (Wako) was added toa final enzyme/protein ratio of 1:200 (w/w) and incubated for 4 h at 37°C. The solution was subsequently diluted with 0.1 M ammoniumbicarbonate(Sigma) to a final concentration below 2 M urea and digested overnightat 37° C. with sequencing-grade modified trypsin (Promega) at aprotein-to-enzyme ratio of 50:1. Peptides were desalted on a C18 Sep-Pakcartridge (Waters) and dried under vacuum. Phosphopeptides were isolatedfrom 2 mg of total peptide mass with TiO₂ as described previously [48].Briefly, dried peptides were dissolved in an 80% acetonitrile (ACN)-2.5%trifluoroacetic acid (TFA) solution saturated with phthalic acid.Peptides were added to the same amount of equilibrated TiO₂ (5-μm beadsize, GL Sciences) in a blocked Mobicol spin column (MoBiTec) that wasincubated for 30 min with end-over-end rotation. The column was washedtwice with the saturated phthalic acid solution, twice with 80% ACN and0.1% TFA, and finally twice with 0.1% TFA. The peptides were eluted witha 0.3 M NH₄OH solution. The pH of the eluates was adjusted to be below2.5 with 5% TFA solution and 2 M HCl. Phosphopeptides were againdesalted with microspin C18 cartridges (Harvard Apparatus).

LC-MS/MS analysis. Chromatographic separation of peptides was carriedout using an EASY nano-LC system (Thermo Fisher Scientific), equippedwith a heated RP-HPLC column (75 μm×45 cm) packed in-house with 1.9 μmC18 resin (Reprosil-AQ Pur, Dr. Maisch). Aliquots of 1 μg totalphosphopeptide sample were analyzed per LC-MS/MS run using a lineargradient ranging from 98% solvent A (0.15% formic acid) and 2% solvent B(98% acetonitrile, 2% water, 0.15% formic acid) to 30% solvent B over120 minutes at a flow rate of 200 nl/min. Mass spectrometry analysis wasperformed on a dual pressure LTQ-Orbitrap mass spectrometer equippedwith a nanoelectrospray ion source (both Thermo Fisher Scientific). EachMS1 scan (acquired in the Orbitrap) was followed by collision-induceddissociation (CID, acquired in the LTQ) of the 20 most abundantprecursor ions with dynamic exclusion for 30 seconds. For phosphopeptideanalysis the 10 most abundant precursor ions were subjected to CID withenabled multistage activation. Total cycle time was approximately 2 s.For MS1, 10⁶ ions were accumulated in the Orbitrap cell over a maximumtime of 300 ms and scanned at a resolution of 60,000 FWHM (at 400 m/z).MS2 scans were acquired using the normal scan mode, a target setting of10⁴ ions, and accumulation time of 25 ms. Singly charged ions and ionswith unassigned charge state were excluded from triggering MS2 events.The normalized collision energy was set to 32%, and one microscan wasacquired for each spectrum.

Label-Free Quantification and Database Searching. The acquired raw-fileswere imported into the Progenesis software tool (Nonlinear Dynamics,Version 4.0) for label-free quantification using the default parameters.MS2 spectra were exported directly from Progenesis in mgf format andsearched using the MASCOT algorithm (Matrix Science, Version 2.4)against a decoy database [49] containing normal and reverse sequences ofthe predicted SwissProt entries of Homo sapiens (world wide web address:ebi.ac.uk, release date May 16, 2012) and commonly observed contaminants(in total 41,250 sequences) generated using the SequenceReverser toolfrom the MaxQuant software (Version 1.0.13.13). To identify proteinsoriginating from Y. enterocolitica, non phosphopeptide enriched sampleswere searched against the same database above including predictedSwissProt entries of Y. enterocolitica (world wide web address:ebi.ac.uk, release date Aug. 15, 2013) The precursor ion tolerance wasset to 10 ppm and fragment ion tolerance was set to 0.6 Da. The searchcriteria were set as follows: full tryptic specificity was required(cleavage after lysine or arginine residues unless followed by proline),2 missed cleavages were allowed, carbamidomethylation (C) was set asfixed modification and phosphorylation (S,T,Y) or oxidation (M) as avariable modification for TiO2 enriched or not enriched samples,respectively. Finally, the database search results were exported as anxml-file and imported back to the Progenesis software for MS1 featureassignment. For phosphopeptide quantification, a csv-file containing theMS1 peak abundances of all detected features was exported and for notenriched samples, a csv-file containing all protein measurements basedon the summed feature intensities of all identified peptides per proteinwas created. Importantly, the Progenesis software was set that proteinsidentified by similar sets of peptides are grouped together and thatonly non-conflicting peptides with specific sequences for singleproteins in the database were employed for protein quantification. Bothfiles were further processed using the in-house developed SafeQuant v1.0R script (unpublished data, available at world wide web address:github.com/eahrne/SafeQuant/). In brief, the software sets theidentification level False Discovery Rate to 1% (based on the number ofdecoy protein sequence database hits) and normalizes the identified MS1peak abundances (Extracted Ion Chromatogram, XIC) across all samples,i.e. the summed XIC of all confidently identified peptide features isscaled to be equal for all LC-MS runs. Next, all quantifiedphosphopeptides/proteins are assigned an abundance ratio for each timepoint, based on the median XIC per time point. The statisticalsignificance of each ratio is given by its q-value (False Discovery Rateadjusted p-values), obtained by calculating modified t-statisticp-values [50] and adjusting for multiple testing [51]. The location ofthe phosphorylated residues was automatically assigned by MASCOT(score >10). All annotated spectra together with the MS raw files andsearch parameters employed, will be deposited to the ProteomeXchangeConsortium (world wide web address: proteomecentral.proteomexchange.org)via the PRIDE partner repository [52]. Sequence alignment was performedusing EMBL-EBI web based ClustalW2 multiple sequence alignment tool atworld wide web address: ebi.ac.ukTools/msa/clustalw2/.

Biodistribution in 4T1 Tumor Allograft Mouse Models

All animal experiments were approved (license 1908; KantonalesVeterinäramt Basel-Stadt) and performed according to local guidelines(Tierschutz-Verordnung, Basel-Stadt) and the Swiss animal protection law(Tierschutz-Gesetz). 6 week old BALB/c mice were ordered from JanvierLabs. After at least one week of accommodation, mice were anesthetizedusing isoflurane and 100 ul 4T1 cells (1×10⁵-1×10⁶ cells) weresubcutaneously injected into the flank of BALB/c mice. Throughout theexperiment, mice were scored for behavior and physical appearance, andsurface temperature, as well as body weight was measured.

Once tumors had developed, mice were administered an 8 mg/ml desferalsolution (10 ml/kg) through i.p. injection. On the following day, micewere infected with Y. enterocolitica MRS40 or Y. enterocolitica MRS40ΔHOPEMT (2×10⁵, 1×10⁶ or 1×10⁷ bacteria) by injection into the tailvein. The inoculum i.v. administered to the mice was validated bydilution plating. In some experiments, tumor progression was followed bydaily measurements of tumor length and width with digital calipers.Tumor volume was determined as 0.523×lenght×width². On respective dayspostinfection, mice were sacrificed by CO₂ inhalation. A blood samplewas immediately isolated through aspiration from the heart. Liver,spleen, lung and the tumor were isolated and their weight determined.The organs and the tumor were homogenized. CFU in each sample wasdetermined by spotting of serial dilutions onto LB agar platescontaining nalidixic acid (35 ug/ml).

B) Results

A Protein Delivery System Based on Type 3 Secretion of YopE FusionProteins

While the very N-terminus of the Y. enterocolitica T3SS effector YopE(SEQ ID No. 1) contains the secretion signal sufficient to translocateheterologous proteins [26], the chaperone-binding site (CBS) for itschaperone (SycE) is not included [53]. We selected the N-terminal 138amino acids of YopE (SEQ ID No. 2) to be fused to proteins to bedelivered, as this had been shown to give best results for translocationof other heterologous T3S substrates [28]. As these N-terminal 138 aminoacids of YopE contain the CBS, we further decided to coexpress SycE. TheSycE-YopE₁₋₁₃₈ fragment cloned from purified Y. enterocolitica pYV40virulence plasmid contains the endogenous promoters of YopE and of itschaperone SycE (FIG. 10 ). Therefore, SycE and any YopE₁₋₁₃₈ fusionprotein are induced by a rapid temperature shift from growth at RT to37° C. Culture time at 37° C. will affect fusion protein amount presentin bacteria. A multiple cloning site (MCS) was added at the 3′ end ofYopE₁₋₁₃₈ (FIG. 10 B) followed by a Myc and a 6×His tag and a Stopcodon.

The background strain was carefully selected. First, to limit thetranslocation of endogenous effectors, we used a Y. enterocoliticastrain that was deleted for all known effectors, Yop H, O, P, E, M and T(named ΔHOPEMT) [54]. In addition, we occasionally used an auxotrophmutant that cannot grow in absence of exogenous meso-2,6-diaminopimelicacid [55]. This strain was deleted for the aspartate-beta-semialdehydedehydrogenase gene (Δasd), and classified as biosafety level 1 by theSwiss safety agency (amendment to A010088/2). In addition, we deletedthe adhesion proteins YadA and/or InvA to offer a larger choice ofbackground strains. While the use of the yadA or yadA/invA strainsreduce the background signalling induced [56], the delivered proteinamount is affected as well [57].

Characterization of YopE Fusion Protein Delivery into Eukaryotic Cells

In an in-vitro secretion assay (see FIG. 1A), protein secretion into thesurrounding liquid is artificially induced. After TCA based proteinprecipitation, Western blot analysis with anti-YopE antibody was used todetermine protein amounts secreted (FIG. 1 B). While a wt strainsecreted full length YopE, the ΔHOPEMT asd strains did not. Uponpresence of YopE₁₋₁₃₈-Myc-His (further termed YopE₁₋₁₃₈-Myc; SEQ ID NO:3) a smaller YopE band became visible (FIG. 1 B). Hence, the YopE₁₋₁₃₈fragment is well secreted in the set up described here. To analyzehomogeneity of protein translocation into eukaryotic cells, we infectedHeLa cells with the YopE₁₋₁₃₈-Myc encoding strain and stained the Myctag by IF (FIGS. 2A and B). While in the beginning only the bacteriawere stained, at 30 min post infection (p.i.) cell outlines start to bevisible, which is enhanced upon increased infection time (FIG. 2 B).This trend is well reflected by the Myc tag staining intensity insideHeLa cells (FIGS. 2A and B). The YopE₁₋₁₃₈-Myc can be detectedeverywhere in the cells (FIG. 2A), except in the nuclei [58].Remarkably, most if not all cells were reached by this approach in acomparable way. As Y. enterocolitica is known to infect many differentcell types [59], we followed YopE₁₋₁₃₈-Myc delivery into various celllines. The same homogenous anti-Myc IF staining was observed in infectedmurine fibroblasts, Jurkat cells and HUVECs (FIG. 11 ). Even more,tuning the MOI up or down allows modulating the protein amount delivered(FIG. 2 C), while still most of the cells remain targeted. A lowbacterial number will not result in few cells with lots of deliveredprotein but rather with most cells containing a low amount of deliveredprotein (FIG. 2 C).

Redirection of T3SS Delivered Proteins to the Nucleus

As YopE itself localized to the cytoplasm (FIG. 2A), it is of specialinterest to test if the YopE₁₋₁₃₈ fragment hampers localization ofnuclear fusion proteins. We therefore added the SV40 NLS to theC-terminus (and N-terminus, similar results) of YopE₁₋₁₃₈-EGFP (SEQ IDNo. 39 and SEQ ID No. 38, respectively). While YopE₁₋₁₃₈-EGFP (SEQ IDNo. 37) led to a weak cytoplasmic staining, YopE₁₋₁₃₈-EGFP-NLS gave riseto a stronger nuclear EGFP signal in HeLa cells infected (FIG. 3 ). Thisindicates that the YopE₁₋₁₃₈ fragment is compatible with the use of anNLS. While mCherry had already been used in plant pathogens [60], thisrepresents a successful delivery of a GFP-like protein via human oranimal pathogenic bacteria encoding a T3SS. This validates the SycE andYopE₁₋₁₃₈ dependent strategy to be very promising for delivery of manyproteins of choice.

Removal of the YopE₁₋₁₃₈ Appendage after Translocation of the FusionProtein to the Eukaryotic Cell

While for bacterial delivery the YopE₁₋₁₃₈ fragment is of great benefit,it might hamper the fusion proteins function and/or localization.Therefore, its removal after protein delivery would be optimal. To thisend, we introduced two TEV cleavage sites (ENLYFQS) (SEQ ID NO: 235)[61-63] in between YopE₁₋₁₃₈ and a fusion partner (the transcriptionalregulator ET1-Myc (SEQ ID No. 36 and 41) [64] and human INK4C (SEQ IDNo. 40 and SEQ ID No. 43)). To keep the advantages of the presentedmethod, we further fused the TEV protease (S219V variant; [65]) toYopE₁₋₁₃₈ (SEQ ID No. 42) in another Y. enterocolitica strain. HeLacells were infected with both strains at once. To allow analysis of thetranslocated fraction of proteins only, infected HeLa cells were lysedat 2 h p.i. (FIG. 4 ) with Digitonin, which is known not to lyse thebacteria ([66]; see FIG. 12 for control). Western blot analysis revealedthe presence of the YopE₁₋₁₃₈-2×TEV-cleavage-site-ET1-Myc orYopE₁₋₁₃₈-2×TEV-cleavage-site-Flag-INK4C-Myc only when cells had beeninfected with the corresponding strain (FIGS. 4A and C). Upon overnightdigestion of this cell-lysate with purified TEV protease, a shifted bandcould be observed (FIGS. 4A and C). This band corresponds to ET1-Myc(FIG. 4 C) or Flag-INK4C (FIG. 4A) with the N-terminal remnants of theTEV cleavage site, most likely only one Serine. Upon coinfection ofcells with the strain delivering the TEV protease, the same cleavedET1-Myc or Flag-INK4C fragment became visible, indicating that the TEVprotease delivered via T3SS is functional and that single cells had beeninfected by both bacterial strains (FIGS. 4A and C). While cleavage isnot complete, the majority of translocated protein is cleaved already 2h post infection and even over-night digestion with purified TEVprotease did not yield better cleavage rates (FIG. 4 B). As reported,TEV protease dependent cleavage might need optimization dependent on thefusion protein [67,68]. TEV protease dependent removal of the YopE₁₋₁₃₈appendage after translocation hence provides for the first time a T3SSprotein delivery of almost native heterologous proteins, changing theamino acid composition by only one N-terminal amino acid.

An alternative approach to the TEV protease dependent cleavage of theYopE fragment consisted in incorporating Ubiquitin into the fusionprotein of interest. Indeed, Ubiquitin is processed at its C-terminus bya group of endogenous Ubiquitin-specific C-terminal proteases(Deubiquitinating enzymes, DUBs). As the cleavage is supposed to happenat the very C-terminus of Ubiquitin (after G76), the protein of interestshould be free of additional amino acid sequence. This method was testedon the YopE1-138-Ubiquitin-Flag-INK4C-MycHis fusion protein. In controlcells infected by YopE1-138-Flag-INK4C-MycHis-expressing bacteria, aband corresponding to YopE1-138-Flag-INK4C-MycHis was found, indicativeof efficient translocation of the fusion protein (FIG. 24 ). When cellswere infected for 1 h withYopE1-138-Ubiquitin-Flag-INK4C-MycHis-expressing bacteria, an additionalband corresponding to the size of Flag-INK4C-MycHis was visible,indicating that part of the fusion protein was cleaved. This resultshows that the introduction of Ubiquitin into the fusion protein enablesto cleave off the YopE1-138 fragment without a need for an exogenousprotease.

Translocation of Type III and Type IV Bacterial Effectors

SopE from Salmonella enterica is a well-characterized guanine nucleotideexchange factor (GEF) that interacts with Cdc42, promoting actincytoskeletal remodeling [69]. Whereas the translocation of YopE₁₋₁₃₈-Mycinto HeLa cells has no effect, translocated YopE₁₋₁₃₈-SopE (SEQ ID No. 5and 135) induced dramatic changes in the actin network (FIG. 5A).Similar results were obtained with another GEF effector protein, IpgB1from Shigella flexneri (SEQ ID No. 4). Remarkably, first changes in theactin cytoskeleton were observed as fast as 2 min p.i. (FIG. 5A).Therefore, one can conclude that T3SS dependent protein delivery happensimmediately after infection is initiated by centrifugation. To proofstrict T3SS dependent transport, one of the T3SS proteins forming thetranslocation pore into the eukaryotic cell membrane was deleted (YopB,see [70]) (FIG. 12 ).

During Salmonella infection, SopE translocation is followed bytranslocation of SptP, which functions as a GTPase activating protein(GAP) for Cdc42 [71]. Whereas the translocation of YopE₁₋₁₃₈-SopE-Myc(SEQ ID No. 135) alone triggered massive F-actin rearrangements, theco-infection with YopE₁₋₁₃₈-SptP (SEQ ID No. 8) expressing bacteriaabolished this effect in a dose dependent manner (FIG. 5 B). An anti-Mycstaining indicated that this inhibition was not due to a reduced levelof YopE₁₋₁₃₈-SopE-Myc translocation (FIG. 5 B). Together these resultsshowed that the co-infection of cells with two bacterial strains is avalid method to deliver two different effectors into single cells toaddress their functional interaction.

The S. flexneri type III effector OspF functions as a phosphothreoninelyase that dephosphorylates MAP kinases p38 and ERK [72]. To test thefunctionality of translocated YopE₁₋₁₃₈-OspF (SEQ ID No. 7), wemonitored the phosphorylation of p38 after stimulation with TNFα. Inuninfected cells or in cells infected with YopE₁₋₁₃₈-Myc expressingbacteria, TNFα□induced p38 phosphorylation. In contrast, aftertranslocation of YopE₁₋₁₃₈-OspF, TNFα-induced phosphorylation wasabolished, showing that the delivered OspF is active towards p38 (FIG.6A).

During Salmonella infection, the type III effector SopB protectsepithelial cells from apoptosis by sustained activation of Akt [73].Whereas the translocation of YopE₁₋₁₃₈-Myc or YopE₁₋₁₃₈-SopE had noeffect on Akt, the translocation of YopE₁₋₁₃₈-SopB (SEQ ID No. 6)induced a strong phosphorylation of Akt at T308 and S473, reflecting theactive form (FIG. 6 B). Similar results were obtained with theSopB-homolog from S. flexneri (IpgD, SEQ ID No. 9). Altogether, ourresults show that the YopE₁₋₁₃₈-based delivery system functions for allT3S effectors tested so far, and that it allows investigating proteinsinvolved in the control of central cellular functions including thecytoskeleton, inflammation and cell survival.

A number of bacteria, including Agrobacterium tumefaciens, Legionellapneumophila and Bartonella henselae, use type IV secretion to injecteffectors into cells. We tested whether the type IV effector BepA fromB. henselae could be translocated into HeLa cells using our tool. Fulllength BepA (SEQ ID No. 10) and BepA_(E305-end) (SEQ ID No. 11)containing the C-terminal Bid domain, were cloned and cells wereinfected with the respective strains. As BepA was shown to induce theproduction of cyclic AMP (cAMP) [74], the level of cAMP in HeLa cellswas measured after infection. Whereas the translocation of the Biddomain of the B. henselae effector BepG (SEQ ID No. 136) failed toinduce cAMP, full length BepA and BepA_(E305-end) triggered cAMPproduction in expected amounts [74] (FIG. 6 C). This result shows, thattype IV effectors can also be effectively delivered by theYopE₁₋₁₃₈-based delivery system into host cell targets and that they arefunctional.

Translocation of Eukaryotic Proteins into Epithelial Cells

To show that human proteins can translocate via type III secretion wefused human apoptosis inducers for delivery by Y. enterocolitica toYopE₁₋₁₃₈ or for delivery by S. enterica to SteA₁₋₂₀, SteA, SopE₁₋₈₁ orSopE₁₋₁₀₅. We then monitored the translocation of the human BH3interacting-domain death agonist (BID, SEQ ID No. 24), which is apro-apoptotic member of the Bcl-2 protein family. It is a mediator ofmitochondrial damage induced by caspase-8 (CASP8). CASP8 cleaves BID,and the truncated BID (tBID, SEQ ID No. 25) translocates to mitochondriawhere it triggers cytochrome c release. The latter leads to theintrinsic mode of caspase 3 (CASP3) activation during which it iscleaved into 17 and 12 kDa subunits [75]. Whereas infection for 1 h withYopE₁₋₁₃₈-Myc or YopE₁₋₁₃₈-BID expressing Y. enterocolitica failed toinduce apoptosis, the translocation of human tBID triggered cell deathin larger extend than the well-characterized apoptosis inducerstaurosporin (FIGS. 7A and C). As expected, the translocation of tBIDlead to the production of CASP3 p17 subunit, even in larger amounts aswith staurosporin (FIG. 7A). To be able to compare translocated proteinamounts to endogenous Bid, HeLa cells were lysed with Digitonin andanalyzed by Western blotting using an anti Bid antibody (FIG. 7 B). T3SSdelivered YopE₁₋₁₃₈-tBID reached about endogenous Bid levels in HeLacells, while delivered YopE₁₋₁₃₈-BID was present in even higherquantities (2.5 fold) (FIG. 7 B). A deep proteome and transcriptomemapping of HeLa cells estimated 4.4 fold 10⁵ copies of BID per singlecell [76]. Therefore, one can conclude that T3SS dependent human proteindelivery reaches 10⁵ to 10⁶ proteins per cell. These numbers fit thecopies per cell of nanobodies translocated via E. coli T3SS [4].Assuming a levelling of a factor of 10 for the MOI and for the durationof the infection, a factor of 3.2 for the time-point of antibioticaddition and for the culture time at 37° C. before infection, thedelivered protein copies/cell can be tuned from some 1000 copies/cell upto some 10⁶ copies/cell Altogether, these results indicated thattranslocated tBID was functional and delivered at relevant levels. Thisvalidated the translocation tool to study the role of proteins in theregulation of apoptosis, a central aspect of cell biology.

We further fused murine tBID (codon optimized for Y. enterocolitica; SEQID No. 194) or the BH3 domains of murine tBID or murine BAX (in bothcases codon optimized for Y. enterocolitica; SEQ ID No. 138 and 139) toYopE₁₋₁₃₈ for delivery by Y. enterocolitica. Whereas infection for 2.5 hwith Y. enterocolitica ΔHOPEMT asd delivering no protein or YopE₁₋₁₃₈-Myc failed to induce apoptosis, the translocation of murine tBID(codon optimized to Y. enterocolitica, SEQ ID No. 194) triggered celldeath in B16F10 (FIG. 16 ), D2A1 (FIG. 17 ), HeLa (FIG. 18 ) and 4T1(FIG. 19 ) cells. The translocation of the BH3 domain of murine BIDcodon optimized for Y. enterocolitica (SEQ ID 138) or murine BAX codonoptimized for Y. enterocolitica (SEQ ID 139) were as well found toinduce massive cell death in B16F10 (FIG. 16 ), D2A1 (FIG. 17 ), HeLa(FIG. 18 ) and 4T1 (FIG. 19 ) cells.

Whereas infection for 4 h with S. enterica aroA bacteria failed toinduce apoptosis, the translocation of murine tBID triggered apoptosis,as the translocation of murine tBID lead to the production of CASP3 p17subunit (FIGS. 20 and 21 ). The extent of apoptosis induction for SopEfusion proteins was larger when using SpiI T3SS inducing conditions(FIG. 20 ), which reflects the transport of SopE exclusively by SpiIT3SS. SteA₁₋₂₀ fused murine tBID failed to induce apoptosis, very likelybecause the secretion signal within the 20 N-terminal amino acids ofSteA is not sufficient to allow delivery of a fusion protein (FIGS. 20and 21 ). Murine tBID fused to full length SteA lead to apoptosisinduction in HeLa cells (FIGS. 20 and 21 ), both in SpiI and SpiII T3SSinducing conditions, reflecting the ability of SteA to be transported byboth T3SS. It has to be noted that even under SpiII T3SS inducingconditions, a partial activity of the SpiI T3SS is expected as seen bythe activity of SopE fusion proteins in SpiII T3SS inducing conditions(FIG. 21 ).

Besides the here functionally elaborated translocated eukaryoticproteins, several other eukaryotic proteins have been secreted using thehere-described tool. This includes for delivery by Y. enterocolitica(FIGS. 13, 14 and 23 ) proteins from cell cycle regulation (Mad2 (SEQ IDNo. 15), CDK1 (SEQ ID No. 14), INK4A (SEQ ID No. 16), INK4B (SEQ ID No.17) and INK4C (SEQ ID No. 18)) as well as parts thereof (INK4A 84-103(SEQ ID No. 158), p107 657-662 (SEQ ID No. 159), p21 141-160 (SEQ ID No.160), p21 145-160 (SEQ ID No. 161), p21 17-33 (SEQ ID No. 162) andcyclin D2 139-147 (SEQ ID No 163)), apoptosis related proteins (Bad (SEQID No. 29), FADD (SEQ ID No. 28), and Caspase 3 p17 (SEQ ID No. 22) andp12 (SEQ ID No. 23), zebrafish Bid (SEQ ID No. 19) and t-Bid (SEQ ID No.20)) as well as parts thereof (tBid BH3 (SEQ ID No.138), Bax BH3 (SEQ IDNo.139)), signalling proteins (murine TRAF6 (SEQ ID No. 12), TIFA (SEQID No. 13)), GPCR Gα subunit (GNA12, shortest isoform, (SEQ ID No. 30)),nanobody (vhhGFP4, (SEQ ID No. 31)) and nanobody fusion constructs fortargeted protein degradation (Slmb-vhhGFP4; (SEQ_ID_Nos. 32, 33, 34)[77]) (FIGS. 13 and 14 ) as well as small GTPases (Rac1 Q61E (SEQ ID No.26 and 137) and RhoA Q63L (SEQ ID No. 27) and Pleckstrin homology domainfrom human Akt (SEQ ID No. 35). Besides the functionally elaboratedapoptosis related proteins (murine tBid, SEQ ID No. 144-147), thisfurther includes for delivery by S. enterica (FIG. 22 ) proteins fromcell cycle regulation (Mad2 (SEQ ID No. 168-169), CDK1 (SEQ ID No.170-171), INK4A (SEQ ID No. 164-165) and INK4C (SEQ ID No. 166-167)).While those proteins have not been functionally validated, thepossibility of T3SS dependent secretion of diverse eukaryotic proteinsin combination with the possible removal of the YopE appendage opens upnew vistas on the broad applicability of T3SS in cell biology andtherapeutic applications.

In Vivo Translocation of Truncated Bid in Zebrafish Embryos InducesApoptosis

An interesting feature of this bacterial tool is the potential use inliving animals. Zebrafish in their embryonic state can be kepttransparent allowing fluorescent staining and microscopy [44,78,79]. Fewzebrafish apoptosis inducers have been described in detail, whereofz-BIM is the most potent [80]. Therefore, we decided to clone z-BIM intoour system. Even if weakly homolgous to human BIM, we assayed thepotency of apoptosis induction of YopE₁₋₁₃₈-z-BIM (SEQ ID No. 21) inhuman epithelial cells. HeLa cells infected for 1 h with the straintranslocating YopE₁₋₁₃₈-z-BIM showed clear signs of cell death. We thenperformed in-vivo experiments with 2 days post fertilization (dpf)zebrafish embryos, using a localized infection model via microinjectionof bacteria into the hindbrain [44]. After infection for 5.5 h the fishwere fixed, permeabilized and stained for presence of CASP3 p17. Uponinfection with the YopE₁₋₁₃₈-Myc expressing strain, bacteria werevisible in the hindbrain region (staining “b”, FIG. 8A I) but noinduction of apoptosis around the bacteria was detected (staining “c”,FIG. 8A I). In contrast, upon infection with the strain deliveringYopE₁₋₁₃₈-z-BIM a strong increase in presence of cleaved CASP3 wasobserved in regions surrounding the bacteria (FIG. 8A II). Automatedimage analysis on maximum intensity z projections confirms thatYopE₁₋₁₃₈-z-BIM translocating bacteria induce apoptosis in nearby cellsby far more than control bacteria do (FIG. 8 B). This indicates thatz-BIM is functional in zebrafish upon bacterial translocation. Theseresults further validate the use of T3SS for eukaryotic protein deliveryin living animals.

Phosphoproteomics Reveal the Global Impact of Translocated Proteins onProtein Phosphorylation

Phosphorylation is a wide-spread post-translational modification whichcan either activate or inactivate biological processes and is thereforea suitable target to study signaling events [81,82]. Despite this, nosystems-level analysis of phosphorylation in apoptosis is availabletoday. To analyze the impact of human tBid delivered into HeLa cells, weused a label-free phosphoproteomic approach by LC-MS/MS. In threeindependent experiments, cells were either left untreated, infected withΔHOPEMT asd+YopE₁₋₁₃₈-Myc or with ΔHOPEMT asd+YopE₁₋₁₃₈-tBid for 30minutes. Cells were lysed, followed by enzymatic digestion,phosphopeptide enrichment and quantification and identification ofindividual phosphopeptides. We compared cells infected with ΔHOPEMTasd+YopE₁₋₁₃₈-Myc to cells infected with ΔHOPEMT asd+YopE₁₋₁₃₈-tBid,allowing us to identify 363 tBid dependent phosphorylation events. 286phosphopeptides showed an increase in phosphorylation whereas 77 wereless phosphorylated upon tBid delivery, corresponding to 243 differentproteins, which we defined as the tBid phosphoproteome. The STRINGdatabase was used to create a protein-protein interaction network of thetBid phosphoproteome [83] (FIG. 9A). Additionally 27 proteins known tobe related to mitochondrial apoptosis were added to the network,building a central cluster. Interestingly, only few proteins from thetBid phosphoproteome are connected to this central cluster indicatingthat many proteins undergo a change in phosphorylation that were so farnot directly linked to apoptotic proteins. To characterize thebiological functions covered by the tBid phosphoproteome, we performed agene ontology analysis using the functional annotation tool of theDatabase for Annotation, Visualization, and Integrated Discovery (DAVID,world wide web address: david.abcc.ncifcrf.gov/) [84,85]. Identifiedbiological functions show that diverse cellular processes are affectedby tBid. Many proteins involved in chromatin rearrangement and theregulation of transcription undergo a change in phosphorylation (i.e.CBX3, CBX5, TRIM28, HDAC1). HDAC1 for example is a histone deacetylaseplaying a role in regulation of transcription. It has been shown thatHDAC1 can modulate transcriptional activity of NF-kB, a protein alsoparticipating in apoptosis. We additionally identified a cluster ofproteins involved in RNA processing which has previously been shown toplay an important role in the regulation of apoptosis [86]. HNRPK forinstance mediates a p53/TP53 response to DNA damage and is necessary forthe induction of apoptosis [87]. Furthermore, the phosphorylation ofproteins involved in protein translation is also affected. Severaleukaryotic initiation factors (i.e. EIF4E2, EIF4B, EIF3A, EIF4G2)undergo a change in phosphorylation, which is in line with theobservation that overall protein synthesis is decreased in apoptoticcells. Interestingly, the phosphorylation of many proteins involved incytoskeleton remodeling (e.g. PXN, MAP1B9 are altered upon tBiddelivery. This is in concordance with the observation that themorphology of cells changes dramatically upon tBid delivery (FIG. 9 B).Cells shrinkage and loss of contact is reflected by the fact that weobserve phosphorylation of adhesion related proteins like ZO2 andPaxillin. Similarly, shrinkage of the nuclei is accompanied byphosphorylation of laminar proteins like LaminA/C and Lamin B1.Altogether, tBID delivery induces a rapid apoptotic response alsoindicated by rupture of the mitochondrial integrity (FIG. 9 B). Weshowed that tBid induced apoptosis affects hundreds of phosphorylationevents participating in diverse cellular processes. While manyidentified proteins have been related to apoptosis, only few were knownto be phosphorylated upon apoptosis induction. The phosphoproteomicapproach thus provides a useful resource for further studies onapoptosis.

Translocation of Eukaryotic Heterologous Fusion Proteins Consisting ofRepeated Identical or Variable Protein Domains into Epithelial Cells

To show that heterologous fusion proteins consisting of repeatedidentical or variable protein domains can translocate via type IIIsecretion we fused murine apoptosis inducers for delivery by Y.enterocolitica to YopE₁₋₁₃₈. As control, we fused murine tBID (codonoptimized for Y. enterocolitica; SEQ ID No. 194) or the BH3 domains ofmurine tBID or murine BAX (in both cases codon optimized for Y.enterocolitica; SEQ ID No. 200 and 201) to YopE₁₋₁₃₈ for delivery by Y.enterocolitica. The heterologous fusion protein consisted in one case ofmurine BH3 domain of tBID fused to itself, resulting inYopE₁₋₁₃₈-(tBID-BH3)₂ (SEQ ID No. 202). In a second case, theheterologous fusion proteins consisted of murine BH3 domain of tBIDfused to murine BH3 domain of BAX, resulting inYopE₁₋₁₃₈-(tBID-BH3)-(BAX-BH3) (SEQ ID No. 203). In the case of murinetBID and murine BAX the codon was optimized for Y. enterocolitica.Schematic representation of repeated identical domains or combination ofdifferent protein domains is shown in FIG. 25 .

Whereas infection for 4 h with Y. enterocolitica ΔHOPEMT asd deliveringYopE ₁₋₁₃₈-Myc failed to induce apoptosis, the translocation of murineBH3 domain tBID (codon optimized to Y. enterocolitica, SEQ ID No. 194)triggered cell death in B16F10 and 4T1 cells (FIGS. 26 and 27 ), with aclear dose-response effect upon increasing multiplicity of infection(MOI). Surprisingly, delivered YopE₁₋₁₃₈-(tBID-BH3)-(BAX-BH3) orYopE₁₋₁₃₈-(tBID-BH3)₂ were found more active than YopE₁₋₁₃₈-(tBID-BH3)at lower MOI (FIGS. 26 and 27 ). This indicates that upon delivery ofrepeated identical domains or combination of different protein domains,the impact on a desired cellular pathway as apoptosis can be enlarged.

Generation of Enhanced Pro-Apoptotic Bacteria

In above mentioned experiments it is shown that the T3SS-based deliveryof pro-apoptotic proteins (e.g. t-BID (SEQ ID No. 25) or BIM (SEQ ID No.21)) efficiently induces cell death in both murine and human cells,including cancerous cells, and that this effect could be increased whenusing murine t-BID optimized to the bacterial codon usage (SEQ ID No.138). This increased cell killing very likely reflects increased amountof protein production and following delivery via T3SS due to optimalcodons used.

In order to optimize the delivery or pro-apoptotic proteins, strainstransformed with different pro-apoptotic proteins have been generatedaccording to Table IV.

TABLE IV Strains transformed with different pro-apoptotic proteinsProtein to Resulting Background be delivred Backbone plasmid Primers.Strain Name strain by T3SS plasmid name Si_Nr.: resistances YopE1-138-Y. enterocolitica YopE1-138- pBad_Si_2 pSi_353 725/726 Nal Amp (Y.enterocolitica ΔyopH, O, P, Y. enterocolitica codon optimized E, M, TΔasd codon optimized murine tBid murine tBid BH3 extended BH3 extendedpart) (by 4 Aa) YopE1-138- Y. enterocolitica YopE1-138- pBad_Si_2pSi_354 727/728 Nal Amp 10 Aa linker- ΔyopH, O, P, 10 Aa linker- (Y.enterocolitica E, M, T Δasd Y. enterocolitica codon optimized codonoptimized murine tBid murine tBid BH3 part) BH3 YopE1-(138- Y.enterocolitica YopE1-138- pSi_357 pSi_374 736/737 Nal Amp Y.enterocolitica ΔyopH, O, P, Y. enterocolitica codon optimized E, M, TΔasd codon optimized murine Bax murine Bax BH3-. BH3 part-enterocolitica Y. enterocolitica codon optimized codon optimized murinetBid murine tBid BH3 BH3 part

Shortening the delivered proteins to the essential domains required forsignaling (SEQ ID No. 138 or 200)) could increase the efficiency of cellkilling (FIG. 28 ). Without being bound by theory, this increase inefficacy is likely to be related to increased amount of proteinproduction and following delivery via T3SS due to smaller size of thedelivered protein. Introduction of a linker between the YopE part andthe BH3 domain of tBID (SEQ ID No. 218) decreased efficacy, as well asextending the BH3 domain by 4 further amino acids (SEQ ID No. 217) (FIG.28 ).

Additionally, synthetic cargos with repeats of such essential domains(e.g. the BH3 domain of t-BID (SEQ ID No. 202))) or combinations ofthese essential domains (e.g. the BH3 domain of t-BID and the BH3 domainof BAX (SEQ ID No. 203 and 219)) were generated. Surprisingly, tandemrepeats of the same or different BH3 domains were found to result inenhanced apoptosis induction on cancerous cell lines (including 4T1 andB16F10 cells, FIG. 28 ). The IC50 (half maximal inhibitoryconcentration), referring to the number of bacteria per eukaryotic cell(MOI) needed in order to kill 50% of such cells, was found to bedecreased upon delivery of tandem repeats of tBID BH3 domain as comparedto a single tBID BH3 domain (FIG. 28 ). This finding was surprising, asthe protein size is increased by fusing as second BH3 domain of t-BID.Due to this, decreased expression and delivery levels of YopE₁₋₁₃₈-(tBIDBH3)₂ (SEQ ID No. 202) as compared to YopE₁₋₁₃₈-tBID BH3 (SEQ ID No. 138or 200) would be expected, and might maximally reach equivalent levels.In order to reach an increase in cell killing activity, the fused tBIDBH3 domains must simultaneously act side by side upon delivery by theT3SS into eukaryotic cells. In case only one tBID BH3 domain in theYopE₁₋₁₃₈-(tBID BH3)₂ construct would be functional, at best the sameefficiency as with YopE₁₋₁₃₈-tBID BH3 might be expected.

In order to increase the genetic stability of YopE₁₋₁₃₈-(tBID BH3)₂ (SEQID No. 202) for in vivo studies, we cloned YopE₁₋₁₃₈-(tBID BH3)₂ (SEQ IDNo. 202) by homologous recombination on the Yersinia virulence plasmidpYV at the native site of YopE and under the native YopE promoter (usingmutator plamids pSI_408 and pSI_419). Such mutators contain the DNAsequence coding for the desired protein, flanked by 200-250 bp ofsequences on both sides corresponding to the site of the respectivegene, where the integration shall take place. These plasmids aretransformed into E. coli Sm10λ pir, from where plasmids were mobilizedinto the corresponding Y. enterocolitica strain. Mutants carrying theintegrated vector were propagated for severeal generations withoutselection pressure. Then sucrose was used to select for clones that havelost the vector. Finally mutants were identified by colony PCR. Theendogenous proteins for the transport by the T3SS (called “Yersiniaouter proteins”, Yops) are encoded by Y. enterocolitica on this 70 kbplasmid, named plasmid of Yersinia Virulence (pYV), which furtherencodes the T3SS apparatus.

Yersinia strains encoding YopE₁₋₁₃₈-(tBID BH3) (SEQ ID No. 138 or 200)or YopE₁₋₁₃₈-(tBID BH3)₂ (SEQ ID No. 202) on the Yersinia virulenceplasmid pYV at the native site of YopE and under the native YopEpromoter were assessed for their capacity of inducing apoptosis incancerous cells (including 4T1 and B16F10 cells, FIG. 29 ). The IC50(half maximal inhibitory concentration), referring to the number ofbacteria per eukaryotic cell (MOI) needed in order to kill 50% of suchcells, was found to be decreased upon delivery of tandem repeats of tBIDBH3 domain as compared to a single tBID BH3 domain, when both proteinsare encoded on the Yersinia virulence plasmid pYV at the native site ofYopE and under the native YopE promoter (FIG. 29 ). This is in agreementwith findings from expression plasmid borne delivery of these proteins(FIG. 28 ). Again, this finding was surprising, as the protein size isincreased by fusing a second BH3 domain of t-BID. Due to this, decreasedexpression and delivery levels of YopE₁₋₁₃₈-(tBID BH3)₂ (SEQ ID No. 202)as compared to YopE₁₋₁₃₈-tBID BH3 (SEQ ID No. 138 or 200) would beexpected, and might maximally reach equivalent levels. In order to reachan increase in cell killing activity, the fused tBID BH3 domains mustsimultaneously act side by side upon delivery by the T3SS intoeukaryotic cells. In case only one tBID BH3 domain in theYopE₁₋₁₃₈-(tBID BH3)₂ construct would be functional, at best the sameefficiency as with YopE₁₋₁₃₈-tBID BH3 might be expected. Furthermore,Yersinia strains encoding YopE₁₋₁₃₈-(tBID BH3)₂ (SEQ ID No. 202) on theYersinia virulence plasmid pYV at the native site of YopE and under thenative YopE promoter were compared for their capacity of inducingapoptosis in cancerous cells to expression plasmid (pBad-MycHisA based)derived delivery of YopE₁₋₁₃₈-(tBID BH3)₂. In agreement with the highercopy number of pBad-MycHisA (20-25 copies) as compared to the pYV (1-6copies are reported), pBad-MycHisA based delivery of YopE₁₋₁₃₈-(tBIDBH3)₂ (SEQ ID No. 202) resulted in a slightly decreased IC50 value on4T1 and B16F10 cells (FIG. 29 ).

Validation of Tumor Specific Growth In Vivo Up to Day 14 Post BacterialAdministration

The experiment of tumor colonization by genetically modified Y.enterocolitica was repeated in a syngeneic murine allograft model (4T1breast cancer model) and bacterial colonization was followed over twoweeks. This time, mice were infected with 1*10⁶ colony forming units(CFU) of Y. enterocolitica ΔyopH,O,P,E,M,T. While obtaining similarresults to the B16F10 model at early days post infection, we couldfurther show that the tumor colonization is consistently found at day 8and up to day 14 after infection (FIG. 30 ). Furthermore, thecolonization remains highly specific with only low counts of bacteriadetected in all other organs assessed (FIG. 31 ). These findingsindicate that Y. enterocolitica ΔyopH,O,P,E,M,T is able to establish apersistent colonization of the tumor thereby preventing clearance by theimmune system.

Efficacy of Y. enterocolitica ΔHOPEMT in Delaying Tumor Progression

In order to assess the impact of YopE₁₋₁₃₈-(tBID BH3)₂ (SEQ ID No. 202)delivered to tumor cells in vivo, we performed studies in wildtypeBalb/C mice allografted s.c. with 4T1 breast cancer cells. We aimed atassessing the Y. enterocolitica ΔHOPEMT strain encoding YopE₁₋₁₃₈-(tBIDBH3)₂ (SEQ ID No. 202) on the Yersinia virulence plasmid pYV at thenative site of YopE and under the native YopE promoter. Mice were i.v.injected with PBS or 1*10⁷ Y. enterocolitica ΔHOPEMT pYV-YopE₁₋₁₃₈-(tBIDBH3)₂, once the tumor had reached a size of 150-250 mm3. The day of thei.v. injection of bacteria was defined as day 0. Tumor volume wasmeasured over the following days (day 0 to day 9 post i.v. injection ofbacteria) with calipers. The tumor volume was normalized to the tumorvolume at day 0 to compensate for any initial heterogeneity in tumorsize. Treatment with Y. enterocolitica ΔHOPEMT pYV-YopE₁₋₁₃₈-(tBID BH3)₂showed an impact on tumor volume progression, with statisticallysignificant tumor reduction at day 8, 9 and 10 post bacterialadministration (FIG. 32 ). Importantly, Y. enterocolitica ΔHOPEMT alonewas found not to impact tumor progression in the 4T1 murine cancer model(FIG. 33 ). These findings highlight that such bacteria and their T3SScan be employed for interference with tumor progression.

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The invention claimed is:
 1. A recombinant Gram-negative bacterialstrain transformed with a vector which comprises in the 5′ to 3′direction: a promoter; a first DNA sequence encoding a delivery signalfrom a bacterial type Ill secretion system (T3SS) effector protein,wherein the delivery signal directs the delivery of a heterologousprotein or domains thereof from the Gram-negative bacterial strain toeukaryotic cells, operably linked to said promoter; and a second DNAsequence encoding: a fusion protein comprising one repetition or severalrepetitions of the same domain of a heterologous protein, wherein thedomain is functional when delivered into eukaryotic cells and whereinthe domain has a molecular weight of between 1-50 kDa, and fused inframe to the 3′ end of said first DNA sequence wherein the heterologousprotein is involved in apoptosis or apoptosis regulation, wherein therecombinant Gram-negative bacterial strain is a Yersinia strain.
 2. Therecombinant Gram-negative bacterial strain of claim 1, wherein saidrecombinant Gram-negative bacterial strain is deficient in producing atleast one T3SS effector protein.
 3. The recombinant Gram-negativebacterial strain of claim 1, wherein the delivery signal from thebacterial T3SS effector protein encoded by the first DNA sequencecomprises the delivery signal from the YopE effector protein or anN-terminal fragment of the delivery signal from the YopE effectorprotein, wherein the fragment of the delivery signal directs thedelivery of a heterologous protein or domains thereof from theGram-negative bacterial strain to eukaryotic cells.
 4. The recombinantGram-negative bacterial strain of claim 1, wherein said Yersinia strainis wild type or deficient in the production of at least one T3SSeffector protein and wherein the delivery signal from the bacterial T3SSeffector protein comprises the N-terminal 138 amino acids of the Y.enterocolitica YopE effector protein which directs the delivery of aheterologous protein or domains thereof from the Gram-negative bacterialstrain to eukaryotic cells.
 5. The recombinant Gram-negative bacterialstrain or the vector of claim 1, wherein the heterologous proteininvolved in apoptosis or apoptosis regulation is selected from the groupconsisting of BH3-only proteins, caspases and intracellular signallingproteins of death receptor control of apoptosis.
 6. The recombinantGram-negative bacterial strain of claim 1, wherein the domain is the BH3domain of apoptosis inducer tBID.
 7. The recombinant Gram-negativebacterial strain of claim 1, wherein the vector comprises a third DNAsequence encoding a protease cleavage site, wherein the third DNAsequence is located between the 3′ end of said first DNA sequence andthe 5′ end of said second DNA sequence.
 8. The recombinant Gram-negativebacterial strain of claim 1, wherein the heterologous protein is apro-apoptotic protein or an anti-apoptotic protein.
 9. The recombinantGram-negative bacterial strain of claim 8, wherein the heterologousprotein is a pro-apoptotic protein selected from the group consisting ofBax, Bak, Diva, Bcl-Xs, Nbk/Bik, Hrk/Dp5, Bmf, Noxa, Puma, Bim, Bad, Bidand tBid, Bok, Apaf1, BNIP1, BNIP3, Bcl-Gs, Beclin 1, Egl-1 and CED-13,Smac/Diablo, FADD, and the Caspase family.
 10. The recombinantGram-negative bacterial strain of claim 8, wherein the heterologousprotein is an anti-apoptotic protein selected from the group consistingof Bcl-2, Bcl-Xl, Bcl-B, Bcl-W, Mcl-1, Ced-9, A1, NR13, IAP family andBfl-1.
 11. The recombinant Gram-negative bacterial strain of claim 1,wherein the domain is a BH3 domain of a heterologous protein involved inapoptosis or apoptosis regulation.
 12. The recombinant Gram-negativebacterial strain of claim 11, wherein the heterologous protein is apro-apoptotic protein or an anti-apoptotic protein.
 13. The recombinantGram-negative bacterial strain of claim 1, wherein the heterologousprotein is a pro-apoptotic protein selected from the group consisting ofof Bax, Bak, Bim, Bad, Bid and tBid.
 14. The recombinant Gram-negativebacterial strain of claim 1, wherein the domain is a BH3 domain ofapoptosis inducer tBID or pro-apoptotic protein Bax.
 15. A recombinantGram-negative bacterial strain transformed with a vector which comprisesin the 5′ to 3′ direction: a first DNA sequence encoding a deliverysignal or a fragment thereof from a bacterial effector protein, whereinthe fragment of the delivery signal from a bacterial effector proteincomprises at least the first 10 and up to 140 amino acids of thedelivery signal and wherein the fragment of the delivery signal directsthe delivery of a heterologous protein or domains thereof from theGram-negative bacterial strain to eukaryotic cells; and a second DNAsequence encoding: a fusion protein comprising one repetition or severalrepetitions of the same domain of a heterologous protein, wherein thedomain is functional when delivered into eukaryotic cells and whereinthe domain has a molecular weight of between 1-50 kDa, and fused inframe to the 3′ end of said first DNA sequence wherein the heterologousprotein is involved in apoptosis or apoptosis regulation, wherein therecombinant Gram-negative bacterial strain is a Yersinia strain.
 16. Avector which comprises in the 5′ to 3′ direction: a promoter, a firstDNA sequence encoding a delivery signal from a bacterial type Illsecretion system (T3SS) effector protein, operably linked to saidpromoter; a second DNA sequence encoding: a fusion protein comprisingone repetition or several repetitions of the same domain of aheterologous protein, wherein the domain is functional when deliveredinto eukaryotic cells and wherein the domain has a molecular weight ofbetween 1-50 kDa, wherein the heterologous protein is involved inapoptosis or apoptosis regulation.
 17. The vector of claim 16, whereinthe domain is the BH3 domain of apoptosis inducer tBID.
 18. The vectorof claim 16, wherein the vector comprises a third DNA sequence encodinga protease cleavage site, wherein the third DNA sequence is locatedbetween the 3′ end of said first DNA sequence and the 5′ end of saidsecond DNA sequence.
 19. A vector which comprises in the 5′ to 3′direction: a first DNA sequence encoding a delivery signal or a fragmentthereof from a bacterial effector protein, wherein the fragment of thedelivery signal from a bacterial effector protein comprises at least thefirst 10 and up to 140 amino acids of the delivery signal and whereinthe fragment of the delivery signal directs the delivery of aheterologous protein or domains thereof from the Gram-negative bacterialstrain to eukaryotic cells; and a second DNA sequence encoding: a fusionprotein comprising one repetition or several repetitions of the samedomain of a heterologous protein, wherein the domain is functional whendelivered into eukaryotic cells and wherein the domain has a molecularweight of between 1-50 kDa, and fused in frame to the 3′ end of saidfirst DNA sequence, wherein the heterologous protein is involved inapoptosis or apoptosis regulation.